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Archive for the ‘Myocardial Ischemia’ Category


A Concise Review of Cardiovascular Biomarkers of Hypertension

Curator: Larry H. Bernstein, MD, FCAP

LPBI

Revised 5/25/2016

 

Introduction

While a large body of work had been done on cholesterol synthesis, HDL and LDL cholesterol, triglycerides, and lipoproteins for a quarter century, and the concept of metabolic syndrome was emerging, there was neither a unifying concept nor a sufficient multivariable approach to apply the use of laboratory markers to clinical practice.  The mathematical foundation for such an evaluation of the biological markers and the computational tools were maturing at the turn of the 20th century, and the interest in outcomes research for improved healthcare practice was maturing. In addition, there was now heavy investment in health information systems that would support emerging health networks of a rapidly consolidating patient base.  This has become important for the pharmaceutical industry and for allied health sciences to enable a suitable method of measuring the effectiveness of drug and of lifestyle changes to improve the population health.

The importance of finding biomarkers for hypertension is significant as stated above. I refer to observations in a lecture by Teresa Seeman, Ph.D., Professor, UCLA Geffen School of Medicine (1).
The missed cased of hypertension in the U.S. alone has been examined by the NHANES studies. Table  I
shows the poor identification of this serious chronic condition. The next table (Table II)*, also from NHANES  (Seeman study) looks at Allostatic Load for biomarkers using component biomarker measurement criterion cutpoints.  Table III* gives the odds ratios for mortality by Allostatic Load Score.

An explanatory problem for our difficulty with diagnosis of a number of hypertension disease “subsets” is that there is peripheral hypertension that might be idiopathic, or it might be related to coexisting diseases with both inflammatory and vascular structural dynamics nature.  In addition, this may be concurrent with pulmonary hypertension, systemic hypertension, and progressive renal disease.  This discussion is reserved for later.  As stated, the late or missed diagnosis of systemic or essential idiopathic hypertension is illustrated in the three Seeman tables (1).

 

Table 1

Table 2

Table 3

 

 

 

 

Table 1*. Missed cases by “self report”

Self-reports

vs undiagnosed

study NHANES 88-94 NHANES 99-2004 NHANES 2005-08
Hypertension %unaware  BP > 140/90 42.7 43.5 39.06
SR-controlled
SR-high

Unaware

  7.45

10

13.88

8.35

10.85

16.12

6.5

10.18

19.98

High cholesterol Chol > 220 g/dl 55.93 49.3 47.05
SR-controlled
SR- high
Unaware
  11.02
8.68
12.12
8.47
8.72
18.5
7.22
8.12
23.46
Diabetes HgA1C > 6.4%      
SR-controlled

SR- high

Unaware

  2.41

3.43

1.64

1.76

5.01

3.09

2.11
5.51
3.09

*modified from Seeman

 

 

 

 

 

 

 

 

 

Table II* USHANES: Allostatic Load – component cutpoints

Biomarker Total N High Risk Percent (%) Cutpoint
DBP (mm Hg) 15,489 1,180   7.62    90
SBP (mm Hg) 15,491 3,461 22.34  140
Pulse Rate 15,117 1,009   6.67    90
HgA1C (%) 15,441 1,482   9.60    6.4
WHR 14,824 6,778 45.72    0.94
HDL Cholesterol (mg/dl) 15,187 3,440 22.65     40
Total Cholesterol

(mg/dl)

15,293 3,196  20.90    240

*From  T. Seaman, UCLA Geffen SOM

 

Table III*. Odds of mortality by Allostatic Load Score.

ALS Odds Ratio
7-8 5
6 2.6
5 2.3
4 2.1
3 1.8
2 1.5
1 1.4

 

*From  T. Seaman, UCLA Geffen SOM

 

I refer to cardiovascular diseases in reference to an aggregate of diseases affecting the heart, the circulatory system from large artery to the capillary, the lungs and kidneys, excluding the lymphatics.
These major disease entities are both separate and interrelated, not necessarily found in the same combinations. However, they account for a growing proportion of illness, apart from cancers, that affect the aging population of western societies. In the discussion that follows, I shall construct a picture of the pathophysiology of cardiovascular diseases, describe the major biomarkers for the assessment of these, point out the relationship of these to hypertension, and try to develop a more targeted approach to the assessment of hypertension and related disorders.

Chronic kidney disease (CKD) is defined as persistent kidney damage accompanied by a reduction in the glomerular filtration rate (GFR) and the presence of albuminuria. The rise in incidence of CKD is attributed to an aging populace and increases in hypertension (HTN), diabetes, and obesity within the U.S. population. CKD is associated with a host of complications including electrolyte imbalances, mineral and bone disorders, anemia, dyslipidemia, and HTN. It is well known that CKD is a risk factor for cardiovascular disease (CVD), and that a reduced GFR and albuminuria are independently associated with an increase in cardiovascular and all-cause mortality.

The relationship between CKD and HTN is cyclic, as CKD can contribute to or cause HTN (3). Elevated BP leads to damage of blood vessels within the kidney, as well as throughout the body. This damage impairs the kidney’s ability to filter fluid and waste from the blood, leading to an increase of fluid volume in the blood—thus causing an increase in BP.

 

A cursory description of the blood circulation

The full circulation involves the heart as a pump, and the arteries and veins, comprising small and large vessels, and capillaries at the point of delivery of oxygen and capture of carbon dioxide, and of transfer of substrates to tissues.  The brain, liver, pancreas and spleen, and endocrines are not further considered here, except for a consideration on neuro-humoral peptides that have emerged in the regulation of blood pressure and are essential to the stress response. The lung and the liver are both important with respect to the exchange of air and metabolites, and both have secondary circulations, the pulmonary and the portal vascular circulations.  In the case of the lungs, the vena cava flows into the right atrium, which delivers unoxygenated blood to the lungs via the right ventricle and right pulmonary artery, which returns to the left atrium by way of the right pulmonary vein.  The blood from the left atrium that flows into the left ventricle is ejected into the aorta.  The coronary arteries that nourish the heart are at the base of the aorta.  The heart muscle is a syncytium, unlike striated muscle, and it is densely packed with mitochondria, suitable for continuous contraction under vasovagal control. This is the anatomical construct, but the physiology is still being clarified because normal function and disease are both a matter of regulatory control.

In order to understand hypertension, we have to view the heart functioning over a long period of time.
In a still frame picture, we envision the left ventricle contracts emptying the oxygenated blood into the circulation. The ejection of blood into the aorta is called systole, by which the blood is delivered by the force of contraction into the circulation.  The filling pressure is called diastole.  So we have a filling and an emptying, and heard by the stethoscope is a lub-dub, synchronously repeated.   A normal systolic blood pressure is below 120. A systolic blood pressure of 120 to 139 means you have prehypertension, or borderline high blood pressure. Even people with prehypertension are at a higher risk of developing heart disease. A systolic blood pressure number of 140 or higher is considered to be hypertension, or high blood pressure. The diastolic blood pressure number or the bottom number indicates the pressure in the arteries when the heart rests between beats. A normal diastolic blood pressure number is less than 80. A diastolic blood pressure between 80 and 89 indicates prehypertension. A diastolic blood pressure number of 90 or higher is considered to be hypertension or high blood pressure. So now we have identified a systolic and a diastolic high blood pressure. Systolic pressure increases with vigorous activity, and becomes normal when the activity resides.  The systolic blood pressure increases with age. Over time, consistently high blood pressure weakens and damages the blood vessels so affected. Moreover, changes in the body’s normal functions may cause high blood pressure, including changes to kidney fluid and salt balances, the renin-angiotensin-aldosterone system, sympathetic nervous system activity, and blood vessel structure and function.

 

Starling’s Law of the Heart

Two principal intrinsic mechanisms, namely the Frank-Starling mechanism and rate induced regulation, enable the myocardium to adapt to changes in hemodynamic conditions. The Frank-Starling mechanism (also referred to as Starling’s law of the heart), is invoked in response to changes in the resting length of the myocardial fibers. Rate-induced regulation is invoked in response to changes in the frequency of the heartbeat.  (3-9).

Frank and Starling (3, 4) showed that an increase in diastolic volume caused an increase in systolic performance. The stretch effect persists across a range of myocardial contractile states, but during exercise it plays only a lesser role augmenting ventricular function maximal exercise. This is because in healthy human subjects adrenergic reflex mechanisms modulate myocardial performance, heart rate, vascular impedance and coronary flow during exercise and changes in these variables can overshadow the effect of fiber stretch or even prevent an increase in end-diastolic volume during stress (5). (See you- tube (6).

According to Lakatta muscle length modulates the extent of myofilament calcium ion (Ca2+) activation (7-9).   Similarly, the fiber length during a contraction, which is determined in part by the load encountered during shortening, also determines the extent of myofilament Ca2+ activation. Therefore, the terms preload, afterload and myocardial contractile state lose part of their significance in light of current knowledge.

 

Biology and High Blood Pressure

Researchers continue to study how various changes in normal body functions cause high blood pressure. The key functions affected in high blood pressure include (10):

Kidney Fluid and Salt Balances

The kidneys normally regulate the body’s salt balance by retaining sodium and water and excreting potassium. Imbalances in this kidney function can expand blood volumes, which can cause high blood pressure.

Renin-Angiotensin-Aldosterone System

The renin-angiotensin-aldosterone system makes angiotensin and aldosterone hormones. Angiotensin narrows or constricts blood vessels, which can lead to an increase in blood pressure. Aldosterone controls how the kidneys balance fluid and salt levels. Increased aldosterone levels or activity may change this kidney function, leading to increased blood volumes and high blood pressure.

Sympathetic Nervous System Activity

The sympathetic nervous system has important functions in blood pressure regulation, including heart rate, blood pressure, and breathing rate. Researchers are investigating whether imbalances in this system cause high blood pressure.

Blood Vessel Structure and Function

Changes in the structure and function of small and large arteries may contribute to high blood pressure. The angiotensin pathway and the immune system may stiffen small and large arteries, which can affect blood pressure.

Two or more types of hypertension

Systemic hypertension

Idiopathic hypertension

Hypertension from chronic renal disease

Pulmonary artery hypertension

Hypertension associated with systemic chronic inflammatory disease (rheumatoid arthritis and other collagen vascular diseases)

Genetic Causes of High Blood Pressure

Much of the understanding of the body systems involved in high blood pressure has come from genetic studies. High blood pressure often runs in families. Years of research have identified many genes and other mutations associated with high blood pressure, some in the renal salt regulatory and renin-angiotensin-aldosterone pathways. However, these known genetic factors only account for 2 to 3 percent of all cases. Emerging research suggests that certain DNA changes during fetal development also may cause the development of high blood pressure later in life.

Environmental Causes of High Blood Pressure

Environmental causes of high blood pressure include unhealthy lifestyle habits, being overweight or obese, and medicines.

Other medical causes of high blood pressure include other medical conditions such as chronic kidney disease, sleep apnea, thyroid problems, or certain tumors.

The common complications of hypertension and their signs and symptoms include:

http://www.nhlbi.nih.gov/health/health-topics/topics/hbp/causes10

 

Pulse Pressure and Stroke Volume

The  pulse pressure is the difference between systolic (the upper number) and diastolic (the lower number) (11).

Systemic pulse pressure = Psystolic – Pdiastolic

The pulse pressure is 40 mmHg for a typical blood pressure reading of 120/80 mmHg.

Pulse pressure (PP) is proportional to stroke volume (SV), the amount of blood pumped from the heart in one beat, and inversely proportional to the compliance or flexibility of the blood vessels, mainly the aorta.

A low (also called narrow) pulse pressure means that not much blood is being expelled from the heart, and can be caused by a number of factors, including severe blood loss due to trauma, congestive heart failure, shock, a narrowing of the valve leading from the heart to the aorta (stenosis), and fluid accumulating around the heart (tamponade).

High (or wide) pulse pressures occur during exercise, as stroke volume increases and the overall resistance to blood flow decreases. It can also occur for many reasons, such as hardening of the arteries (which can have numerous causes), various deficiencies in the aorta (mainly) or other arteries, including leaksfistulas, and a usually-congenital condition known as AVM, pain/anxiety, fever, anemia, pregnancy, and more. Certain medications for high blood pressure can widen pulse pressure, while others narrow it. A chronic increase in pulse pressure is a risk factor for heart disease, and can lead to the type of arrhythmia called atrial fibrillation or A-Fib.

 

Hypertension Background and Definition

The prevalence of CKD has steadily increased over the past two decades, and was reported to affect over 13% of the U.S. population in 2004.  In 2009, more than 570,000 people in the United States were classified as having end-stage renal disease (ESRD), including nearly 400,000 dialysis patients and over 17,000 transplant recipients.  A patient is determined to have ESRD when he or she requires replacement therapy, including dialysis or kidney transplantation. A National Health Examination Survey (NHANES) spanning 2005-2006 showed that 29% of US adults 18 years of age and older were hypertensive, and of those with high blood pressure (BP), 78% were aware they were hypertensive, 68% were being treated with antihypertensive agents, and only 64% of treated individuals had controlled hypertension (12, 13). In addition, data from NHANES 1999-2006 estimated that 30% of adults 20 years of age and older have prehypertension, defined as an untreated SBP of 120-139 mm Hg or untreated DBP of 80-89 mmHg (12, 13).

Hypertension is the most important modifiable risk factor for coronary heart disease (the leading cause of death in North America), stroke (the third leading cause), congestive heart failure, end-stage renal disease, and peripheral vascular disease. The 2010 Institute for Clinical Systems Improvement (ICSI) guideline (14) on the diagnosis and treatment of hypertension indicates that systolic blood pressure (SBP) should be the major factor to detect, evaluate, and treat hypertension In adults aged 50 years and older. The 2013 joint European Society of Hypertension (ESH) (15) and the European Society of Cardiology (ESC) (16) guidelines recommend that ambulatory blood-pressure monitoring (ABPM) be incorporated into the assessment of cardiovascular risk factors and hypertension.

The JNC 7 (17) identifies the following as major cardiovascular risk factors:

  • Hypertension: component of metabolic syndrome
  • Tobacco use, particularly cigarettes, including chewing tobacco
  • Elevated LDL cholesterol (or total cholesterol ≥240 mg/dL) or low HDL cholesterol: component of metabolic syndrome
  • Diabetes mellitus: component of metabolic syndrome
  • Obesity (BMI ≥30 kg/m 2): component of metabolic syndrome
  • Age greater than 55 years for men or greater than 65 years for women: increased risk begins at the respective ages; the Adult Treatment Panel III used earlier age cut points to suggest the need for earlier action
  • Estimated glomerular filtration rate less than 60 mL/min
  • Microalbuminuria
  • Family history of premature cardiovascular disease (men < 55 years; women < 65 years)
  • Lack of exercise

The Eighth Report of the JNC (JNC 8), released in December 2013 no longer recommends just thiazide-type diuretics as initial therapy in most patients. In essence, the JNC 8 recommends treating to 150/90 mm Hg in patients over age 60 years; for everybody else, the goal BP is 140/90 (18).

Biomarkers Associated with Hypertension

The biomarkers associated with hypertension are for the most part derived from features that characterize the disordered physiology. We might first consider the measurement of blood pressure. Then it becomes necessary to analyze the physiological elements that largely contribute to blood pressure. Finally, there are several biomarkers that have loomed large as measures are myocardial function or myocardial cell death, and are also not independent of renal function, that are indicators of short term and long term cardiovascular status. Having already indicated the importance of measurement of pulse, diastolic and systolic blood pressure in the routine examination of physical status, which is related to cardiac output we shall pay attention to the pulse pressure and pulse wave velocity.    These were defined in the preceding discussion.  They are critically related to the development of hypertension and in the long term, they emerge significantly earlier than either congestive heart failure, chronic kidney disease, acute coronary syndrome, stroke, or cardio-renal syndrome.

Even though cardiovascular disease (CVD), the leading cause of death in developed countries, is not predicted by classic risk factors, there are elements of the risk factor association that need further exploration and will be dissected, such as activity level, obesity, lipids, diabetes mellitus, family history and stress.  Further analysis will point to endocrine and/or metabolic factors that drive cardiovascular risk.

In taking into account the blood pressure measurements, we consider the pulse pressure (PP) and the pulse wave velocity (PWV).  If we refer back to the stroke volume and the Law of the Heart, the systolic blood pressure (SBP) is increased with increased left ventricular output that raises the left ventricular (LV) afterload. This coincides with a decrease in diastolic pressure (DBP) that accompanies a change in coronary artery perfusion (CAP).  Thus, many studies point to increased SBP as a strong risk factor for stroke and CVD.  However, there are sufficient studies that indicate the brachial artery pulse pressure (PP) is a strong determinant of CVD and stroke, and these two elements, SBP and brachial artery PP, may be an indicator of increased arterial stiffness in hypertensive patients and the general population. Brachial PP is also a determinant of recurrent events after acute coronary syndrome (ACS) or with left ventricular hypertrophy (LVH), or the risk of CHF in the aging population, and of all-cause-mortality in the general population.  In addition, the aortic PWV calculated from the Framingham equations was a suitable predictor of CVD risk. In a classic study of arterial stiffness and of CVD and all-cause mortality in an essential hypertension cohort at the Broussais Hospital between 1980 and 1996 (19), the carotid-femoral PWV was measured as an indicator of aortic stiffness, and it was found to be significantly associated with all-cause and CVD mortality independent of previous CVD, age, and diabetes. They tested the hypothesis that aortic stiffness is a predictor of cardiovascular and all-cause mortality in hypertensive patients based on the consideration that the elastic properties of the aorta and central arteries are the major determinants of systemic arterial impedance, and the PWV measured along the aortic and aorto-iliac pathway is the most clinically relevant. They assessed arterial stiffness by measuring the PWV using  the Moens-Korteweg equation based on the increase of the square root of the elasticity modulus in stiffer arteries (20).

PWV as a Diagnostic Test

To assess the performance of PWV considered as a diagnostic test, with the use of receiver operating characteristic (ROC) curves, they calculated sensitivities, specificities, positive predictive values, and negative predictive values of PWV at different cutoff values, first to detect the presence of AA in the overall population and second to detect patients with high 10-year cardiovascular mortality risk in the subgroup of 462 patients without AA with age range from 30 to 74 years. Optimal cutoff values of PWV were defined as the maximization of the sum of sensitivity and specificity.

The main finding of the study was that PWV was a strong predictor of cardiovascular risks as determined by the Framingham equations in a population of treated or untreated subjects with essential hypertension (21). They measured the PWV from foot-to-foot transit time in the aorta for a noninvasive evaluation of regional aortic stiffness, which allows an estimate of the distance traveled by the pulse. The presence of a PWV > 13 m/s, taken alone, appeared as a strong predictor of cardiovascular mortality with high performance values (21). Their work and other studies (22, 23) established increased pulse pressure, the major hemodynamic consequence of increased aortic PWV, as a strong independent predictor of cardiac mortality, mainly MI, in populations of normotensive and hypertensive subjects.

In addition to the findings above, the PWV was found to be an independent predictor of future increase in SBP and of incident hypertension in the Baltimore study (21). The authors reported that in a subset of 306 subjects who were normotensive at baseline, hypertension developed in 105 (34%) during a median follow-up of 4.3 years (range 2 to 12 years). PWV was also an independent predictor of incident hypertension (hazard ratio 1.10 per 1 m/s increase in PWV, 95% confidence interval 1.00 to 1.30, p = 0.03) in individuals with a follow-up duration greater than the median. The authors (21) concluded that carotid-femoral PWV measured using nondirectional transcutaneous Doppler probes (model 810A, 9 to 10-Mhz probes, Parks Medical Electronics, Inc., Aloha, Oregon) could be done to identify normotensive individuals who should be targeted for the implementation of interventions aimed at preventing or delaying the progression of subclinical arterial stiffening and the onset of hypertension.  They reported that age, BMI, and MAP were independently associated with higher SBP on the last visit (Table IV); in addition, PWV was also independently associated with higher SBP on the last visit, and explained 4% of its variance. As shown in Table V, age, BMI, and MAP (p = 0.09, p = 0.009, p < 0.0001 respectively for the interaction terms with time) were predictors of the longitudinal changes in SBP. In addition, PWV was also an independent predictor of the longitudinal increase in SBP (p = 0.003 for the interaction term with time).

In addition, they report that in the group with follow-up duration greater than the median (in which all subjects remained normotensive for the first 4.3 years), beyond age (hazard ratio [HR] 1.02 per 1 year, 95% confidence interval [CI] 0.99 to 1.04, p = 0.2) and SBP (HR 1.05 per 1 mm Hg, 95% CI 1.01 to 1.09, p = 0.006), both HDL (HR 0.96 per 1 mg/dl, 95% CI 0.93 to 0.99, p = 0.02) and PWV (HR 1.10 per 1 m/s, 95% CI 1.00 to 1.30, p = 0.03) (Fig. 1) were independent predictors of incident HTN.

Their findings in a longitudinal projection indicate that PWV, a marker of central arterial stiffening, is an independent determinant of longitudinal SBP increase in healthy BLSA volunteers, and an independent risk factor for incident hypertension among normotensive subjects followed up for longer than 4 years. The study was accompanied by a commentary in the same journal that states: “Pulse wave velocity (PWV) is a simple measure of the time taken by the pressure wave to travel over a specific distance. By virtue of its intrinsic relation to the mechanical properties of the artery by the Moens–Kortweg formula (PWV=√(Eh/2)Rρ; where E is the Young’s Modulus of the arterial wall, h the wall thickness, R the end- diastolic radius and ρ is the density of blood)(20), and buoyed a number of longitudinal studies that reported on the independent predictive value of PWV measurement for cardiovascular events and mortality in various populations, PWV is now widely accepted as the ‘gold standard’ measure of arterial stiffness.

 

 

 

Table IV Multiple Regression Analysis Evaluating the Predictors of Last Visit SBP 21

Variable Parameter
Estimate
Standard
Error
p Value
Age (yrs) 0.32 0.06 <0.0001
Gender (men) 0.65 1.78 0.71
Race (white) −1.22 2.00 0.54
Smoking (ever) 2.48 1.61 0.12
BMI (kg/m2)* 0.61 0.22 0.006
MAP (mm Hg)* 0.60 0.08 <0.0001
PWV (m/s)* 1.56 0.38 <0.0001
Heart rate (beats/min) 0.08 0.06 0.20
Total cholesterol (mg/dl) −0.005 0.02 0.83
Triglycerides (mg/dl) −0.009 0.01 0.50
HDL cholesterol (mg/dl) −0.001 0.07 0.98
Glucose (mg/dl) −0.02 0.06 0.75

 

 

 

 

 

 

 

 

Table V Predictors of Longitudinal SBP Derived From a Linear Mixed-Effects Regression Model 21

Variable Coefficient Standardized

Coefficient

95% Confidence

Interval

p Value
Time (yrs) 3.14 0.14 0.61 to 5.66 0.02
Age (yrs) −0.37 0.25 −0.68 to −0.06 0.02
Age2 (yrs2)* 0.006 0.08 0.002 to 0.008 <0.0001
Gender (men) 0.61 0.03 −1.26 to 2.47 0.52
BMI (kg/m2)* 0.25 0.11 −0.01 to 0.50 0.06
MAP (mmHg)* 1.03 0.47 0.93 to 1.12 <0.0001
PWV (m/s) 0.29 0.12 −0.16 to 0.74 0.21
Time × age* 0.02 0.04 −0.002 to 0.038 0.09
Time × BMI* 0.10 0.06 0.02 to 0.183 0.009
Time × MAP* −0.08 −0.12 −0.11 to −0.05 <0.0001
Time × PWV* 0.22 0.08 0.07 to 0.36 0.003

 

 

Figure 1 21

http://content.onlinejacc.org/data/Journals/JAC/23115/10065_gr1.jpeg

Figure 2.21

http://content.onlinejacc.org/data/Journals/JAC/23115/10065_gr2.jpeg

The interest in this physiological measure is illustrated by the increasing number and diversity of research publications in this arena related to human hypertension, relating PWV to pathophysiological processes (for example, homocysteine, inflammation and extracellular matrix turnover and disorders related to hypertension, such as sleep apnea). The epidemiology, genetic associations and prognostic implications of PWV (and arterial stiffness) have also been reported as has the relationship to hemodynamics, cardiac structure and function.” (24) Furthermore, arterial stiffening may be “characterized by an increase in (central) PP and changes in the morphology of the arterial waveform, both of which can now be measured non-invasively using tonometers from commercially available devices. Wave reflection is typically characterized by aortic pressure augmentation (ΔP) and the augmentation index (ΔP/PP) (Figure 3)(24). Higher augmented pressure, as an index of wave reflection, has been linked to adverse clinical outcomes in different populations.

Figure 3.24

Analysis of the pressure waveform. The initial systolic pressure is labelled as P1 and augmented pressure ( P) is typically measured as the difference between peak pressure (P2) and P1. Augmentation index is  P/PP. PP, pulse pressure.    http://www.nature.com/jhh/journal/v22/n10/images/jhh200847f1.gif 24

A review by Payne et al. (25) states that aortic stiffness and arterial pulse wave reflections determine elevated central systolic pressure and are associated with risk of adverse cardiovascular outcomes. This is because an impaired compensatory mechanism through matrix metalloproteinases of remodeling to compensate for changes in wall stress, possibly related to angiotensin II and inhibition of the vascular adhesion protein semicarbazide-sensitive amine oxidase, related to reduced elastin fiber cross-linking. This has implications for pharmacological agents that target age-related advanced glycation end-product cross-links. This also brings into consideration NO playing a considerable role. But they caution that the endogenous NO synthase inhibitors asymmetric dimethylarginine and L-NG-monomethyl arginine associated with clinical atherosclerosis don’t appear to be associated with arterial stiffening. The matter leaves much to be explained.  The mechanisms underlying arterial stiffness could well require insights into inflammation, calcification, vascular growth and remodeling, and endothelial dysfunction. Nevertheless, arterial stiffness is independently associated with cardiovascular outcome in most of the situations where it has been examined.  Given this train of thinking, O’Rourke (26) considers a progressive arterial dilatation with repeated cycles of stress that leads to degeneration of the arterial wall and increases the pressure wave impulse and wave velocity, augmenting the pressure in late systole. Drugs may reduce wave reflection, but have no direct effect on arterial stiffness.  However, reduction in wave reflection decreases aortic systolic pressure augmentation.  DK Arnett (26) depicts the effect of persistently elevated blood pressure in the following diagram (Figure 4).

 

Figure 4.26  Both transient and sustained stiffening of the artery are likely to be present in hypertension.

An initial elevation in blood pressure may establish a positive feedback in which hypertension biomechanically increases arterial stiffness without any structural change. This elevated blood pressure   might later lead to additional vascular hypertrophy and hyperplasia, collagen deposition, and atherosclerosis, and fixed elevations in arterial stiffness.  As to a genetic factor, she refers to a gene contributing to pulse pressure on chromosome 8 located at 32 cM, which also contains the lipoprotein lipase (LPL) gene which has been associated with hypertension. LPL may be an important candidate gene for pulse pressure.  She specifically identifies a relationship between genetic regions contributing to aortic compliance in African American sibships ascertained for hypertension in Figure 5 (27).  These results suggest there may be influential genetic regions contributing to aortic compliance in African American sibships ascertained for hypertension (27). Collectively, these two studies, the first to our knowledge, indicate the presence of genetic factors influencing hypertension.

Other authors state that PWV has a direct relationship to intrinsic elasticity of the arterial wall, and it is an independent predictor of CVD related morbidity and mortality, but it is not associated with classical risk factors for atherosclerosis (28).  They point out that PWV doesn’t increase during early stages of atherosclerosis, as measured by intima-media thickness and non-calcified atheroma, but it does increase in the presence of aortic calcification that occurs with advanced atherosclerotic plaque. Age-related
PWV measurement. Carotid-to-femoral PWV is calculated by dividing the distance (d) between the two arterial sites by the difference in time of pressure wave arrival between the carotid (t1) and femoral artery (t2) referenced to the R wave of the electrocardiogram.

Figure 5. Linkage of arterial compliance on chromosome 2: HyperGEN27

Widening of the pulse pressure is the major cause of age-related increase in prevalence of hypertension and is related to arterial stiffening. (28)  Commonly used points for measuring the PWV are the carotid and femoral artery because they are superficial and easy to access. Arterial distensibility is measured by the Bramwell and Hill equation (29): PWV = √(V × ΔP/ρ × ΔV), where ρ is blood density. This is shown in Figure 6.

 

Figure 6 28

 

View larger version:

 

Furthermore, these authors (28) report arterial stiffness increases with age by approximately 0.1 m/s/y in East Asian populations with low prevalences of atherosclerosis, but some authors have found accelerated stiffening between 50 and 60 years of age. In contrast, stiffness of peripheral arteries increases less or not at all with increasing age. Again, ageing of the arterial media is associated with increased expression of matrix metalloproteinases (MMP), which are members of the zinc-dependent endopeptidase family and are involved in degradation of vascular elastin and collagen fibers. Several different types of MMP exist in the vascular wall, but in relation to arterial stiffness, much interest has focused on MMP-2 and MMP-9.  This concludes the discussion of PP and PWV in the evolution of hypertension.

 

Diagnostic Biomarkers of essential hypertension.

Ioannidis and Tzoulaki (30) reviewed the literature on 10 popular ‘‘new’’ biomarkers and found that each one had accrued more than 6000 publications.1 The predictive effects of these popular blood biomarkers for coronary heart disease in the general population are listed in Table VI (31).

 

Table VI.* Predictive Value of New Biomarkers 30,31

Biomarker Adjusted Relative Risk (95% C.I.)
Triglycerides 0.99 (0.94–1.05)
C-reactive protein 1.39 (1.32–1.47)
Fibrinogen 1.45 (1.34–1.57)
Interleukin 6 1.27 (1.19–1.35)
BNP or NT-proBNP 1.42 (1.24–1.63)
Serum albumin 1.2 (1.1–1.3)
ICAM-1 (0.75–1.64)
Homocysteine 1.05 (1.03–1.07)
Uric acid 1.09 (1.03–1.16)

*Ionnidis and Tzoulaki from Giles
The majority of these biomarkers show small effects, if any, even in combination.  Giles (31) points out that an elevated homocysteine level might be of great importance to a young person with a myocardial infarction and a positive family history of similar occurrences. Emerging biomarkers, eg, asymmetric and symmetric dimethylarginine and galectin-3, are promising more specific biomarkers based on pathophysiologies for cardiovascular disease. Even then, blood pressure remains the biomarker par excellence for hypertension and for many other cardiovascular entities.

The importance of blood pressure was highlighted by the report of the cardiovascular lifetime risk pooling project.(10) Starting at 55 years of age, 61,585 men and women were followed over an average of 14 years, ie, 700,000 person-years. Individuals who maintained or decreased their blood pressure to normal levels had the lowest remaining lifetime risk for cardiovascular disease (22–41%) compared with individuals who had or developed hypertension by 55 years of age (42–69%). The study indicated that efforts should continue to emphasize the importance of lowering blood pressure and avoiding or delaying the incidence of hypertension to reduce the lifetime risk for cardiovascular disease

A small study involving 120 hypertensive patients with or without heart failure tried to establish a multi-biomarker approach to heart failure (HF) in hypertensive patients using N-terminal pro BNP (32). The following biomarkers were included in the study: Collagen III N-terminal propeptide (PIIINP), cystatin C (CysC), lipocalin-2/NGAL, syndecan-4, tumor necrosis factor-α (TNF-α), interleukin 1 receptor type I (IL1R1), galectin-3, cardiotrophin-1 (CT-1), transforming growth factor β (TGF-β) and N-terminal pro-brain natriuretic peptide (NT-proBNP). The highest discriminative value for HF was observed for NT-proBNP (area under the receiver operating characteristic curve (AUC) = 0.873) and TGF-β (AUC = 0.878). On the basis of ROC curve analysis they found that CT-1 > 152 pg/mL, TGF-β < 7.7 ng/mL, syndecan > 2.3 ng/mL, NT-proBNP > 332.5 pg/mL, CysC > 1 mg/L and NGAL > 39.9 ng/mL were significant predictors of overt HF. There was only a small improvement in predictive ability of the multi-biomarker panel including the four biomarkers with the best performance in the detection of HF (NT-proBNP, TGF-β, CT-1, CysC) compared to the panel with NT-proBNP, TGF-β and CT-1 (absent  CysC). The biomarkers with different pathophysiological backgrounds (NT-proBNP, TGF-β, CT-1) give additive prognostic value for incident compared to NT-proBNP alone.

Inflammation has been associated with pathophysiology of hypertension and vascular damage. Resistant hypertensive patients (RHTN) have unfavorable prognosis due to poor blood pressure control and higher prevalence of target organ damage. Endothelial dysfunction and arterial stiffness are involved in such condition. Previous studies showed that RHTN patients have higher arterial stiffness and endothelial dysfunction than controlled hypertensive and normotensive subjects. The relationship between high blood pressure levels and arterial stiffness may be explained in part, by inflammatory pathways. Previous studies also found that hypertensive subjects have higher levels of inflammatory cytokines including TNF-α, IL-10, IL-1β and CRP. Moreover, IL-1β correlates with arterial stiffness and levels of blood pressure, which are particularly high in patients with resistant hypertension. Increased inflammatory cytokines levels might be related to the development of vascular damage and to the higher cardiovascular risk of resistant hypertensive patients. Elevated BP may cause cardiovascular structural and functional alterations leading to organ damage such as left ventricular hypertrophy, arterial and renal dysfunction. TNF-α inhibition reduced systolic BP and endothelial inflammation in SHR [33]. They also found that IL-1β correlates with arterial stiffness and levels of blood pressure, even after adjust for age and glucose [33]. These investigators then demonstrated that isoprostane levels, an oxidative stress marker, were associated with endothelial dysfunction in these patients [33].

Chao et al. carried out studies of kallistatin (34-36). Kallistatin is an endogenous protein in human plasma as a tissue Kallikrein-Binding Protein (KBP). Tissue kallikrein is a serine protease that releases vasodilating kinin peptides from kininogen substrate. The tissue kallikrein-kinin system is involved in mediating beneficial effects in hypertension as well as cardiac, cerebral and renal injury. KBP was later identified as a serine protease inhibitor (serpin) because of its ability to inhibit tissue kallikrein activity, and was subsequently named “kallistatin”. Kallistatin is mainly expressed in the liver, but is also present in the heart, kidney and blood vessel. Kallistatin protein contains two structural elements: an active site and a heparin-binding domain. The active site of kallistatin is crucial for complex formation with tissue kallikrein, and thus tissue kallikrein inhibition.

Kallistatin is expressed in tissues relevant to cardiovascular function, and has consequently been shown to have vasodilating properties.  Kallistatin has pleiotropic effects in vasodilation and inhibition of inflammation, angiogenesis, oxidative stress, fibrosis, and cancer progression. Injection of a neutralizing Kallistatin antibody into hypertensive rats aggravates cardiovascular and renal injury in association with increased inflammation, oxidative stress and tissue remodeling.  Neither the blood pressure-lowering effect nor the vasorelaxation ability of kallistatin is abolished by icatibant (Hoe140, a kinin B2 receptor antagonist), indicating that kallistatin-mediated vasodilation is unrelated to the tissue kallikrein-kinin system.

The findings reported indicate that kallistatin exerts beneficial effects against hypertension and organ damage. Kallistatin levels in circulation, body fluids or tissues were lower in patients with liver disease, septic syndrome, diabetic retinopathy, severe pneumonia, inflammatory bowel disease, and cancer of the colon and prostate. In addition, reduced plasma kallistatin levels are associated with adiposity and metabolic risk in apparently healthy African American youths. Considered a negative acute-phase protein, circulating kallistatin levels as well as hepatic expression are rapidly reduced within 24 hours after Lipopolysaccharide (LPS) induced endotoxemia in mice. Similarly, circulating kallistatin levels are markedly decreased in patients with septic syndrome and liver disease. Taking together, the studies indicate that kallistatin exhibits potent anti-inflammatory activity.

The pathogenesis of hypertension and cardiovascular and renal diseases is tightly linked to increased oxidative stress and reduced NO bioavailability (37-39). Time-dependent elevation of circulating oxygen species are associated with reduced kallistatin levels in animal models of hypertension and cardiovascular and renal injury. Stimulation of NO formation by kallistatin may lead to inhibition of oxidative stress and thus multi-organ damage. On the other hand, endogenous kallistatin depletion by neutralizing antibody increased oxidative stress and aggravated cardiovascular and renal damage.

A human kallistatin gene polymorphism has been shown to correlate with a decreased risk of developing acute kidney injury during septic shock. Kallistatin levels are markedly reduced in both humans and mice with sepsis syndrome. However, kallistatin administration protects against lethality and organ injury in animal models of toxic septic shock. Moreover, kallistatin levels are decreased in patients with liver disease, septic shock, inflammatory bowel disease, severe pneumonia and acute respiratory distress syndrome. Taken together, the results indicate that kallistatin has the potential to be a molecular biomarker for patients with sepsis, cardiovascular and metabolic disorders.

Pulmonary hypertension (PH) is defined as a mean pulmonary artery pressure of .25 mmHg at rest or .30 mmHg with exercise. Right heart catheterization is required for the definitive diagnosis. Subsequent investigations are instituted to further characterize the disease. The 6-min walk test (6MWT), a measure of exercise capacity, and the New York Heart Association (NYHA)/World Health Organization (WHO) functional classification, a measure of severity, are used to follow the clinical course while receiving treatment, and these both correlate with disease severity and prognosis (43).

Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary vasculature that leads to exercise limitation, right heart failure, and death. There is a need for biomarkers that can aid in early detection, disease surveillance, and treatment monitoring in PAH. Several potential molecules have been investigated; however, only brain natriuretic peptide is currently recommended at diagnosis and for follow-up of PAH patients.

ANP is released from storage granules in atrial tissue, while BNP is secreted from ventricular tissue in a constitutive fashion. ANP secretion is stimulated by atrial stretch caused by atrial volume overload; BNP is released in response to ventricular stretch. Natriuretic peptides act on the kidney, causing natriuresis and diuresis, and relax vascular smooth muscle, causing arterial and venous dilatation, leading to reduced blood pressure and ventricular preload. ANP and BNP are released as prohormones and then cleaved into the active peptide and an inactive N-terminal fragment (43).

Natriuretic peptide precursors are released in response to atrial and ventricular stretch, cleaved into active molecules and inactive precursors and convert guanosine 59-triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), leading to their various physiological actions.

There are a number of confounding factors in the interpretation of natriuretic peptide levels, including left heart disease, sex, age and renal dysfunction. Since most studies exclude patients with left heart disease and renal dysfunction, it becomes problematic extrapolating these results to an unselected population (43).

Endothelin-1 (ET-1) is a peptide found in abundance in the human lung and, through action of endothelin receptors (ETA and ETB) on vascular smooth muscle cells, is implicated in the pathogenesis of PAH. Endothelin receptor antagonists are approved for the treatment of PAH. Levels of circulating ET-1 and related molecules are logical biomarkers of interest in PAH. ET-1 is elevated in PAH compared to controls, and correlates with pulmonary hemodynamic parameters. In addition, higher ET-1 levels are associated with increased mortality in patients treated for PAH. ET-1’s precursor, big-ET-1, has a longer half-life and hence is more stable than ET-1.

Endothelin-1 ET-1 is a potent endogenous vasoconstrictor and proliferative cytokine. The ET-1 gene is translated to prepro-ET-1 which is then cleaved, by the action of an intracellular endopeptidase, to form the biologically inactive big ET-1. ET-converting enzymes further cleave this to form functional ET-1 . There are two ET receptor isoforms, termed type A (ETA), located predominantly on vascular smooth muscle cells, and type B (ETB), predominantly expressed on vascular endothelial cells but also on arterial smooth muscle. Activation of both receptor subtypes, when located on vascular smooth muscle, results in vasoconstriction and cell proliferation. In addition, the endothelial ETB receptor mediates vasodilatation and clearance of ET-1 (43).

Prepro-ET-1 is cleaved to inactive big ET-1 and then further cleaved to form active ET-1. This acts on vascular smooth muscle via the ETA and ETB receptors, causing vasoconstriction and cell proliferation, and on endothelial cells via ETB receptors, releasing nitric oxide (NO) and prostacyclin (PGI2), causing vasorelaxation.

As a biomarker, ADMA has been evaluated in several different classes of PH (43, 44). In IPAH, plasma levels are significantly higher than in healthy, matched controls. In such patients, plasma ADMA correlates positively with right atrial pressure, and negatively with mixed venous oxygen saturation, stroke volume, cardiac index and survival. On stepwise multiple regression analysis, ADMA is an independent predictor of mortality and, using Kaplan–Meier survival curves, patients with supramedian ADMA levels have significantly worse survival than those with inframedian levels.

Patients with idiopathic PAH, plasma levels of Ang-1 and Ang-2 were higher in PAH patients as compared to healthy controls.  Moreover, higher plasma levels of Ang-2 were associated with lower CI and mixed venous oxygen saturation (SvO2) and higher PVR, and, with therapy initiation, changes in Ang-2 correlated with changes in hemodynamics (45, 46).

Endostatin is an antiangiogenic peptide. It is synthesized by myocardium, is detectable in the peripheral circulation of patients with decompensated heart failure, and predicts mortality.48 In PAH, reduced RV myocardial oxygen delivery is felt to contribute to a transition from RV adaptation to failure (46).

Cyclic guanosine monophosphate (cGMP) is an intracellular second messenger of nitric oxide and an indirect marker of natriuretic peptide production (46).

Human pentraxin 3 (PTX3) is a protein synthesized by vascular cells that regulates angiogenesis, inflammation, and cell proliferation (46).

N-terminal propeptide of procollagen III (PIIINP), carboxy-terminal telopeptide of collagen I (CITP), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase I (TIMP-1)(46).

Osteopontin (OPN) is a matricellular protein that mediates cell migration, adhesion, remodeling, and survival of the vascular and inflammatory cells (46).

F2-isoprostane is a marker of lipid peroxidation of arachidonic acid, which stimulates endothelial cell proliferation and ET-1 synthesis and may play a role in the pathogenesis of PAH (46).

Circulating fibrocytes are bone marrow-derived cells (CD45 /collagen I ) that contribute to organ fibrosis and extracellular matrix deposition (46).

Circulating miRs (46)

Despite many other substances being investigated as potential biomarkers in PAH, more research is needed to validate the results of small studies and assess their clinical utility. Widespread clinical use of current investigational biomarkers will require validated clinical laboratory techniques and increased knowledge of levels in the healthy population as well as other disease states.

Here are important tests in clinical practice (47):

 

6-min walk distance

Cardiac index

WHO FC

PIIINP

Higher tertiles associated with worse disease

worse renal function

higher right atrial pressure (RAP)

CITP – vascular remodeling

 

Recent guidelines (17, 18) encourage the use of screening examinations, such as an echocardiogram (UCG), in high-risk populations for the early detection of PAH . To detect PAH in patients with connective tissue disease (CTD), the obvious screening tests are an UCG and spirometry, including assessment of the diffusing capacity of the lung for carbon monoxide (DLCO). Previous studies have suggested that B-type natriuretic peptide (BNP) and its N-terminal prohormone (NT-proBNP) are potential biomarkers for PAH. However, neither BNP nor NT-pro BNP are specific biomarkers of the degeneration of the pulmonary artery; rather, they are biomarkers of cardiac burden resulting from right heart failure.

Human pentraxin 3 (PTX3) is a specific biomarker for PAH, reflecting pulmonary vascular proteins. They are divided into short and long pentraxins on the basis of their primary structure.
C-Reactive protein (CRP) and serum amyloid P are the classic short pentraxins that are produced in the liver in response to systemic inflammatory cytokines (48). In contrast, PTX3 is one of the long pentraxins. It is synthesized by local vascular cells, such as smooth muscle cells, endothelial cells and fibroblasts, as well as innate immunity cells at sites of inflammation. PTX3 plays a key role in the regulation of cell proliferation and angiogenesis (49).

Increased plasma PTX3 levels have been reported in patients with acute myocardial injury in the
24 h after admission to hospital, with levels returning to normal after 3 days. Similarly, PTX3 levels are higher in patients with unstable angina pectoris, with the changes in PTX3 levels found to be independent of other coronary risk factors, such as obesity and diabetes mellitus. Finally, high serum PTX3 levels have been reported in patents with vasculitis, such as small-vessel vasculitis  and Takayasu aortitis.

Mean plasma PTX3 concentrations in the CTD-PAH and CTD patients were 5.02+0.69 ng/mL (range 1.82–12.94 ng/mL) and 2.40+0.14 ng/mL (range 0.70–4.29 ng/mL), respectively (Table 2). Log transformation of the data revealed significantly higher PTX3 levels in CTD-PAH than in CTD patients (1.49+0.12 vs. 0.82+0.06 log ng/mL, respectively; P = 0.001).(not shown)(50)

Figure 1. Serum pentraxin 3 (PTX3) concentrations in 50 patients with pulmonary arterial hypertension (PAH) and 100 healthy controls, and their correlation with serum concentrations of other biomarkers. A: Comparison of PTX3 concentrations in PAH patients and healthy controls. Mean plasma PTX3 concentrations were 4.4060.37 and 1.94+0.09 ng/mL in the controls and PAH patients, respectively. B: Distribution of log-transformed PTX3 concentrations in PAH patients and healthy controls. C: Log-transformed PTX3 concentrations were significantly higher in patients with PAH than in healthy controls (1.34+0.07 vs. 0.55+0.05 log ng/mL, respectively; P,0.001). D, E: There was no correlation between plasma concentrations of PTX3 and either B-type natriuretic peptide (BNP; r=0.33, P=0.02) or C-reactive protein (CRP; r=0.21, P=0.14) in PAH patients. (not shown) (50)

 

Table 2. Clinical characteristics and biomarkers in patients with connective tissue disease, with or without pulmonary arterial hypertension.

CTD-PAH ( n =17)                CTD alone ( n =34)       P -value

Age (years)                                 56.3+4.6                                 56.3+2.7               0.990

No. women (%)                         15 (88)                                      31(91)                  0.745

No. with SSc (%)                       10 (59)                                      20 (59)                    1

No. with heart failure (%)          1 (6)                                         0                            –

No. being treated for PAH (%)   17 (100)                                  0                           –

Serum PTX3 (mg/dL)                   5.02+0.69                          2.40+0.14             0.001

Serum CRP (mg/dL)                   0.24+0.09                            0.22+0.04             0.936

Serum BNP (pg/mL)                 189.3+74.                            4 49.3+12.1            0.014

…..  CTD, connective tissue disease; PAH, pulmonary arterial hypertension; SSc, scleroderma;

Figure 3. Receiver operating characteristic (ROC) curves for pentraxin 3 (PTX3) and other biomarkers in patients with connective tissue disease (CTD). The areas under the ROC curve (AUCROC) for PTX3 was 0.866 (95% confidence interval (CI) 0.757–0.974). The star indicates the threshold concentration of 2.85 ng/mL PTX3 that maximized true-positive and false-negative results (sensitivity 94.1%, specificity 73.5%). The AUCROC for C-reactive protein (CRP) was 0.518 (95% CI 0.333–0.704), whereas that for B-type natriuretic peptide (BNP) was 0.670 (95% CI 0.497–0.842). (50)  http://dx.doi.org:/10.1371/journal.pone.0045834.g003

This study was to determine whether PTX3, the regulation of which is independent of that of the systemic inflammatory marker CRP, is a useful biomarker for diagnosing PAH. The investigators found that PTX3 may be a more sensitive biomarker for PAH than BNP, which is, to date, the most established biomarker for PAH, especially in patients with CTD-PAH. Their findings suggest that PTX3 does not reflect the cardiac burden due to the pulmonary hypertension, but rather the activity of pulmonary vascular degeneration because PTX3 levels were significantly decreased after active treatment specifically for PAH (50). PLoS ONE 7(9): e45834. http://dx.doi.org:/10.1371/journal.pone.0045834.

Pharmacologic treatment for pulmonary arterial hypertension (PAH) remains suboptimal and mortality rates are still high, even with pulmonary vasodilator therapy. In addition, we have only an incomplete understanding of the pathobiology of PAH, which is characterized at the tissue level by fibrosis, hypertrophy and plexiform remodeling of the distal pulmonary arterioles. Novel therapeutic approaches that might target pulmonary vascular remodeling, rather than pulmonary vaso-reactivity, require precise patient phenotyping both in terms of clinical status and disease subtype. However, current risk stratification models are cumbersome and not precise enough for choosing or assessing the results of therapeutic intervention. Biomarkers used in patients with left heart failure, such as troponin-T and N-terminal pro-B-type natriuretic peptide (NT-proBNP) are elevated in PAH patients but tend to simply reflect increased circulating plasma volumes and elevated right heart pressure, rather than conveying information about disease mechanism.

In this issue of Heart, Calvier and colleagues (see page 390) (51)propose galectin-3 as a useful biomarker in PAH. The rationale for this hypothesis is that elevated aldosterone levels induce an increase in serum levels of galectin-3, a β-galactoside-binding lectin expressed by circulating myocytes, endothelial cells and other cardiovascular cell types. Among other effects, activation of the aldosterone/galactin-3 pathway promotes fibrosis (51), suggesting that elevated levels will correlate with the severity of PAH due to increased pulmonary arteriolar remodeling. To test this hypothesis, serum levels were measured in a total of 57 patients – 41 with idiopathic PAH (iPAH) and 16 with PAH associated with a connective tissue disorder (CTD). The magnitude of elevation in serum levels of aldosterone, galectin-3 and NT-proBNP each correlated with the severity of PAH. However, as shown in figure 1, although serum levels of galectin-3 were elevated in both iPAH and PAH-CTD patients, aldosterone was elevated only in those with iPAH.

In addition, elevated vascular cell adhesion molecule 1 (VCAM-1) and proinflammatory, anti-angiogenic interleukin 12 (IL-12) in were elevated only in PAH-CTD patients, not in those in iPAH. These data suggest that aldosterone and galectin-3 can be used as biomarkers “in tandem” that reflect both the severity and cause of PAH (52).

In the accompanying editorial, Maron (see page 335) summarizes the knowledge gaps in PAH and concludes: “Taken together, Calvier and colleagues provide a key contribution to an underdeveloped area of pulmonary vascular medicine and in doing so identify galectin-3/aldosterone as promising biomarker(s) for informing both disease pathobiology and clinical status in PAH. The rationale of this pursuit in PAH was based, in part, on lessons earned from left heart failure in which the importance of systemically circulating vasoactive factors to clinical trajectory is well established. In this regard, the current work not only develops a novel scientific avenue worthy of further investigation, but also adds to the evolving body of evidence implicating a role for neurohumoral activation in the pathophysiology of PAH”.

Rheumatoid arthritis (RA) affects about 1% of the population and is known to be a significant risk factor for cardiovascular disease, with a 3-fold increased risk of myocardial infarction, a 2-fold increased risk of sudden death and a 50% increase in cardiovascular mortality rates. However, outcomes after PCI in RA patients have not been well characterized and there is little data on the possible effects of disease modifying therapy for RA on risk of restenosis after percutaneous coronary intervention (PCI). In a single center retrospective cohort study, Sintek and colleagues (53)(see page 363) compared the primary endpoint of repeat target vessel revascularization (TVR) in 143 RA patients matched to 541 other.

Pathophysiological targets of differing imaging modalities, demonstrate targets for tracers/contrast agents/pharmacotherapy used in SPECT, PET, MRI and echocardiography to assess myocardial viability.  (Not shown. Adapted from Schuster et al., J Am Coll Cardiol 2012; 59:359–70.)

Ischemic cardiomyopathy implies significant left ventricular systolic dysfunction with an underlying pathophysiology that includes myocardial scarring, hibernation and stunning, or a combination of these disease states. The role of imaging in assessment of myocardial viability is emphasized (not shown) (54) with brief summaries of the role of echocardiography, single photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI). The effects of revascularization in patients with ischemic cardiomyopathy remain controversial. Instead, the key elements of evidence based therapy for ischemic cardiomyopathy are standard medical therapy for heart failure combined with implantable cardiac defibrillation (ICD) and/or biventricular pacing device therapy in appropriate patients.

The relationship between the heart and the kidney in hypertension and heart failure

Hypertension is undoubtedly a factor in the treatment of chronic kidney disease because of the relationship between kidney function and BP components that have been studied in people with CKD, diabetes, and hypertension.  Cystatin C was used to evaluate the association between kidney function and both SBP and DBP and 24-h creatinine clearance (CrCl) among 906 participants in the Heart and Soul Study.  (56).  The study investigators hypothesized that although both creatinine and cystatin C are freely filtered at the glomerulus, a major difference between them is that creatinine is secreted by renal tubules, whereas cystatin C is metabolized by the proximal tubule and only a small fraction appears in the urine. In addition, Cystatin C has also been shown to be a stronger predictor of adverse outcomes than serum creatinine. Based on the more linear relationship of cystatin C with GFR, they hypothesized that cystatin C would have a stronger association with SBP than conventional measures of kidney function. Their results found that SBP was linearly associated with cystatin C concentrations (1.19 ± 0.55 mm Hg increase per 0.4 mg/L cystatin C, P = .03) across the range of kidney functions, but only in subjects with CrCl <60 mL/min (6.4 ± 2.13 mm Hg increase per 28 mL/min, P = .003), not >60 mL/min. Further, the DBP was not associated with cystatin C or CrCl. However, PP was linearly associated with both cystatin C (1.28 ± 0.55 mm Hg per 0.4 mg/L cystatin, P = .02) and CrCl <60 mL/min (7.27 ± 2.16 mm Hg per 28 mL/min, P = .001). The relationship between SBP and cystatin C by decile is shown in Figure 7 and Table 3.

Figure 7.

Mean systolic blood pressure (SBP) and diastolic blood pressure (DBP) by decile of kidney function measured as cystatin C. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2771570/bin/nihms-153474-f0001.jpg

 

 

Table 3

Linear regression of systolic blood pressure by kidney function (N = 906)

Age-adjusted Multivariable adjusted*
Measure N β coefficient P β coefficient P
Cystatin-C (per 0.4 mg/L [SD] increase) 1.75 ± 0.72 .01 1.19 ± 0.55 .03
    Overall
    >1.0 551 2.23 ± 0.07 .03 1.23 ± 0.03 .04
    <1.0 355 1.59 ± 0.04 .71 0.54 ± 0.01 .87
Spline P value for difference in slopes .85
24-h CrCl (per 28 mL/min [SD] decrease)
    Overall 1.96 ± 0.76 .01 0.91 ± 0.61 .14
    <60 222 11.20 ± 2.74 <.001 6.40 ± 2.13 .003
    >60 684 0.31 ± 0.99 .42 0.36 ± 0.77 .64
    Spline P-value for difference in slopes .01

The results for both Cystatin C and for eGFR are in agreement with incidence rates for heart failure (57)categorized by ejection fraction (EF) and kidney function over 1992−2000 in the Cardiovascular Health Study. Estimated glomerular filtration rate (mL/min per 1.73 m2) is labeled as “eGFR”. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2258307/bin/nihms-39968-f0002.jpg).

The association of cystatin C with risk for SHF appeared linear across quartiles of cystatin C (57) and slightly stronger at the highest categories of cystatin C, whereas the lower three quartiles of cystatin C had similar risks for DHF. Participants with an estimated GFR ≥ 60 mL/min per 1.73 m2 had an equal likelihood of developing DHF or SHF, whereas participants with an estimated GFR < 60 mL/min per 1.73 m2 had a greater likelihood of developing SHF.

When an interaction term for HF type (SHF or DHF) was inserted into a fully adjusted standard Cox proportional hazards model with HF with either type of EF as the outcome, the association of continuous cystatin C with SHF was significantly greater than the association of cystatin C with DHF ( P value for interaction < 0.001). The association of estimated GFR and SHF compared with DHF was weaker (P value for interaction = 0.06 for the fully adjusted model).

Ascending quartiles of cystatin C were associated with increasing adjusted risk for the development of “unclassified” HF, defined by the absence of a point-of-care EF measurement. The magnitude of the fully adjusted hazard ratios for the association between cystatin C and risk of unclassified HF were intermediate between those described for DHF and SHF [hazard ratios (95% confidence intervals) for each higher quartile of cystatin C 1.00 (reference), 1.12 (0.80−1.57), 1.84 (1.34−2.51), 2.18 (1.58−3.00)]. The authors state that increased left atrial filling pressures trigger the release of atrial natriuretic peptide and inhibition of vasopressin, which leads to decreased renal sympathetic tone and diuresis early in the pathogenesis of HF (57).  They suggest that even relatively small decrements in k58idney function contribute to the risk of SHF.

Aldosterone plays a key role in homeostatic control and maintenance of blood pressure (BP) by regulation of extracellular volume, vascular tone, and cardiac output. Taking this assumption further, a study unrelated to that above explored the magnitude of the effect of relative aldosterone excess in predicting peripheral as well as aortic blood pressure in a cohort of patients undergoing coronary angiography.  (58) They found that mean peripheral systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the entire cohort were 141 ± 24 mm Hg and 81 ± 11 mm Hg, respectively. Median SBP and aortic SBP increased steadily and significantly from aldosterone/renin ratio (ARR), respectively; p < 0.0001 for both) after multivariate adjustment for parameters potentially influencing BP. ARR emerged as the second most significant independent predictor (after age) of mean SBP and as the most important predictor of mean DBP in this patient cohort.  The authors stress the importance of the ARR in modulating BP over a much wider range than is currently appreciated, as it was already known that the ARR was positively associated with pulse wave velocity in young normotensive healthy adults, indicating that relative aldosterone excess might affect arterial remodeling and precede BP rise as a result of increased vascular stiffness. In this study the ARR was calculated as the PAC/PRC ratio (pg/ml/pg/ml). An ARR >50 pg/ml had a sensitivity and specificity of ARR of 89% and 96%, respectively, for primary aldosteronism. The ARR was modeled as a continuous ratio (with log-transformed values).  The study carried out a multivariate stepwise regression analysis for predictors of BP (not shown). They illustrate (not shown) that marked increases in PRC are a major characteristic of lower ARR categories, and that  across a broad range of ARR values, inappropriately elevated aldosterone levels exert a strong effect on BP values and constitute the most important and second-most important predictor of DBP and SBP, respectively.

Cystatin C may be ordered when a health practitioner is not satisfied with the results of other tests, such as a creatinine or creatinine clearance, or wants to check for early kidney dysfunction, particularly in the elderly, and/or wants to monitor known impairment over time. In diverse populations it has been found to improve the estimate of GFR when combined in an equation with blood creatinine. A high level in the blood corresponds to a decreased glomerular filtration rate (GFR) and hence to kidney dysfunction. Since cystatin C is produced throughout the body at a constant rate and removed and broken down by the kidneys, it should remain at a steady level in the blood if the kidneys are working efficiently and the GFR is normal.

Chronic kidney disease (CKD) is defined as the presence of: persistent and usually progressive reduction in GFR (GFR <60 mL/min/1.73 m2) and/or albuminuria (>30 mg of urinary albumin per gram of urinary creatinine), regardless of GFR. Cystatin C is an index of GFR, especially in patients where serum creatinine may be misleading (eg, very obese, elderly, or malnourished patients); for such patients, use of CKD-EPI cystatin C equation is recommended to estimate GFR. Cystatin C eGFR may have advantages over creatinine eGFR in certain patient groups in whom muscle mass is abnormally high or low (for example quadriplegics, very elderly, or malnourished individuals). Blood levels of cystatin C also equilibrate more quickly than creatinine, and therefore, serum cystatin C may be more accurate than serum creatinine when kidney function is rapidly changing (59) (for example amongst hospitalized individuals).

It is a low molecular weight (13,250 kD) cysteine proteinase inhibitor that is produced by all nucleated cells and found in body fluids, including serum. Since it is formed at a constant rate and freely filtered by the kidneys, its serum concentration is inversely correlated with the glomerular filtration rate (GFR); that is, high values indicate low GFRs while lower values indicate higher GFRs, similar to creatinine. While both cystatin C and creatinine are freely filtered by glomeruli, cystatin C is reabsorbed and metabolized by proximal renal tubules. Thus, under normal conditions, cystatin C does not enter the final excreted urine to any significant degree, and the serum concentration is unaffected by infections, inflammatory or neoplastic states, or by body mass, diet, or drugs.  GFR can be estimated (eGFR) from serum cystatin C utilizing an equation which includes the age and gender of the patient (CKD-EPI cystatin C equation, developed by Inker et al. (59) It demonstrated good correlation with measured iothalamate clearance in patients with all common causes of kidney disease, including kidney transplant recipients.

According to the National Kidney Foundation Kidney Disease Outcome Quality Initiative (K/DOQI) classification, among patients with CKD, irrespective of diagnosis, the stage of disease should be assigned based on the level of kidney function:

Table 4

Stage Description GFR mL/min/BSA
1 Kidney damage with normal or  increased GFR 90
2 Kidney damage with mild decrease in  GFR 60-89
3 Moderate decrease in GFR 30-59
4 Severe decrease in GFR 15-29
5 Kidney failure <15 (or dialysis)

(http://www2.kidney.org/professionals/kdoqi/guidelines_ckd/p4_class_g1.htm)

In a study to evaluate cystatin C as a measure of renal function in comparison to serum creatinine, 500 patients had cystatin C measured by nephelometry and glomerular filtration rate (GFR) measured by nonradiolabeled iothalamate clearance (59). In addition, serum creatinine was measured and the patients’ medical records reviewed. The correlation of 1/cystatin C with GFR (r=0.90) was significantly superior than 1/creatinine (r=0.82, p<0.05) with GFR. The superior correlation of 1/cystatin C with GFR was observed in the various clinical subgroups of patients studied (ie, subjects with no suspected renal disease, renal transplant patients, recipients of some other transplant, patients with glomerular disease, and patients with non-glomerular renal disease). The findings indicated that cystatin C may be superior to serum creatinine for the assessment of GFR in a wide spectrum of patients (59). Others have similarly found that cystatin C correlates better than serum creatinine for assessment of GFR. (60)

Patients were screened for 3 chronic kidney disease (CKD) studies in the United States (n = 2,980) and a clinical population in Paris, France (n = 438)(61).   GFR was measured by using urinary clearance of iodine125-iothalamate in the US studies and chromium51-EDTA in the Paris study. GFR was calculated using the 4 new equations based on serum cystatin C alone, serum cystatin C, serum creatinine, or both with age, sex, and race. New equations were developed by using linear regression with log GFR as the outcome in two thirds of data from US studies. Internal validation was performed in the remaining one third of data from US CKD studies; external validation was performed in the Paris study.

Mean mGFR, serum creatinine, and serum cystatin C values were 48 mL/min/1.73 m2 (5th to 95th percentile, 15 to 95), 2.1 mg/dL, and 1.8 mg/L, respectively. For the new equations, coefficients for age, sex, and race were significant in the equation with serum cystatin C, but 2- to 4-fold smaller than in the equation with serum creatinine (62, 63). Measures of performance in new equations were consistent across the development and internal and external validation data sets. Percentages of estimated GFR within 30% of mGFR for equations based on serum cystatin C alone, serum cystatin C, serum creatinine, or both levels with age, sex, and race were 81%, 83%, 85%, and 89%, respectively. The equation using serum cystatin C level alone yields estimates with small biases in age, sex, and race subgroups, which are improved in equations including these variables. It is concluded that Serum cystatin C level alone provides GFR estimates not linked to muscle mass, and that an equation including serum cystatin C level in combination with serum creatinine level, age, sex, and race provides the most accurate estimates.
The authors report that absence of urinary excretion has made it difficult to rigorously evaluate cystatin C as a filtration marker and to examine its non-GFR determinants. They also point out that a high level of variation in the cystatin C assay (64, 65), and standardization and calibration of clinical laboratories will be important to obtain accurate GFR estimation using cystatin C, as has been shown for creatinine.

The study reported above was followed by a major study by Inker LA, et al. (59). Their findings are summarized as follows. Mean measured GFRs were 68 and 70 ml per minute per 1.73 m2 of body-surface area in the development and validation data sets, respectively. In the validation data set, the creatinine–cystatin C equation performed better than equations that used creatinine or cystatin C alone. Bias was similar among the three equations, with a median difference between measured and estimated GFR of 3.9 ml per minute per 1.73 m2 with the combined equation, as compared with 3.7 and 3.4 ml per minute per 1.73 m2 with the creatinine equation and the cystatin C equation (P=0.07 and P=0.05), respectively. Precision was improved with the combined equation (interquartile range of the difference, 13.4 vs. 15.4 and 16.4 ml per minute per 1.73 m2, respectively [P=0.001 and P<0.001]), and the results were more accurate (percentage of estimates that were >30% of measured GFR, 8.5 vs. 12.8 and 14.1, respectively [P<0.001 for both comparisons]). In participants whose estimated GFR based on creatinine was 45 to 74 ml per minute per 1.73 m2, the combined equation improved the classification of measured GFR as either less than 60 ml per minute per 1.73 m2 or greater than or equal to 60 ml per minute per 1.73 m2 (net reclassification index, 19.4% [P<0.001]) and correctly reclassified 16.9% of those with an estimated GFR of 45 to 59 ml per minute per 1.73 m2 as having a GFR of 60 ml or higher per minute per 1.73 m2.

Other studies have established the importance of cystatin C levels(66, 67) and the factors influencing cystatin C levels on renal function measurement (68), including an implication that cystatin C, an alternative measure of kidney function, was a stronger predictor of the risk of cardiovascular events and death than either creatinine or the estimated GFR (69). This includes the Dallas Heart Study (30) finding that cystatin C was independently associated with a specific cardiac phenotype of concentric hypertrophy, including increased LV mass, concentricity, and wall thickness, but it was not associated with LV systolic function or volume. This association was particularly robust in hypertensives and blacks. The Cystatin C concentrations within stages of CKD are shown in Table 5 (70).

Table 5

      Cystatin C level
Stage a Description GFR range a (ml/min/1.73 m2) Native kidney disease b Transplant recipient c
1 Normal or increased GFR 90 0.80 0.87
2 Mildly decreased GFR 60 to 89 0.80 to 1.09 0.87 to 1.23
3 Moderately decreased GFR 30 to 59 1.10 to 1.86 1.24 to 2.24
4 Severely decreased GFR 15 to 29 1.87 to 3.17 2.25 to 4.10
5 Kidney Failure <15 >3.17 >4.10

a GFR estimates and CKD stage will be inaccurate if there is a calibration difference with the Dade-Behring BN II Nephelometer assay used in this study.

b Using the prediction equation: GFR=66.8 (cystatin C)-1.30.

c Using the prediction equation: GFR=76.6 (cystatin C)-1.16.

 

Copeptin, a novel marker

Urinary albumin excretion is a powerful predictor of progressive cardiovascular and renal disease. Copeptin is the inactive C-terminal fragment of the vasopressin precursor. It is a reliable marker of vasopressin secretion serves as a useful substitute for circulating vasopressin concentration. This allows  for the indirect measurement of vasopressin in epidemiological studies. Moreover, it has been shown that copeptin is a candidate biomarker for pneumonia 32), a predictor of outcome in heart failure, and is a powerful predictor of renal disease associated with albumin excretion (71).  Figure 8 shows the association between copeptin and 24-hour urinary volume, 24-h urinary osmolality and osmolality (71).

 

Figure 8

 

Association between quintiles of copeptin and median 24-h UAE (upper panel) and prevalence of microalbuminuria (lower panel) for males and females. Differences between the quintiles were tested by Kruskal–Wallis test. UAE, urinary albumin excretion.

 

 

Table 6 shows the association between copeptin concentration and urinary albumin excretion (UAE) in a log-log plot (71).

 

Model Corrected for β 95% CI for β P
Males        
 1 − (Crude) 0.25 0.20–0.30 <0.001
 2 As 1+age 0.21 0.16–0.26 <0.001
 3 As 2+MAP, BMI, smoking, glucose, cholesterol, CRP, and eGFR 0.10 0.05–0.16 <0.001
 4 As 3+diuretics and ACEi/ARB. 0.09 0.04–0.15 0.001
         
Females
 1 − (Crude) 0.19 0.15–0.23 <0.001
 2 As 1+age 0.17 0.14–0.22 <0.001
 3 As 2+MAP, BMI, smoking, glucose, cholesterol, CRP, and eGFR 0.16 0.11–0.21 <0.001
 4 As 3+diuretics and ACEi/ARB. 0.17 0.12–0.21 <0.001

ACEi, angiotensin-converting enzyme inhibitor; ARB, angiotensin-II-receptor blocker; BMI, body mass index; CHD, coronary heart disease; CI, confidence interval; CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; MAP, mean arterial pressure.

Log copeptin concentration was entered in the regression analyses as independent and log UAE as the dependent variable. Copeptin was associated with UAE in all age groups, but this association is the strongest when subjects are older. Twenty-four-hour urinary volume and 24-h urinary osmolarity were significantly different, with 24-h urinary volume being higher and 24-h urinary osmolarity being lower in the oldest age group when compared with the youngest age group. In both males and females, high copeptin concentration (a surrogate for vasopressin) is associated with low 24-h urinary volume and high 24-h urinary osmolarity. However, urinary osmolarity was independently associated with UAE, but it was weaker than that between copeptin and UAE.  This might indicate that induction of specific glomerular hyperfiltration or decreased tubular albumin reabsorption are associated with this relationship. In addition, subjects with higher levels of copeptin had lower renal function.  These investigators concluded that copeptin (a reliable substitute for vasopressin) is associated with UAE and microalbuminuria, consistent with the hypothesis that vasopressin induces UAE (72).  Other studies indicated that copeptin levels are increased in patients with pulmonary artery hypertension (73), and
higher serum copeptin levels, a surrogate for arginine vasopressin (AVP) release, are associated not only with systolic and diastolic blood pressure but also with several components of metabolic syndrome (74) including obesity, elevated concentration of triglycerides, albuminuria, and serum uric acid level.

 

 

Natriuretic peptides in the evaluation of heart failure

The brain type natriuretic peptide (BNP) and the N-terminal pro B-type natriuretic peptide (NT proBNP), but not yet the atrial natriuretic peptide have gained prominence in the evaluation of patients with CHF, which may be with or without preserved ejection fraction . Richards et al. (75)  make the following points.

 

  • Threshold values of B-type natriuretic peptide (BNP) and N-terminal prohormone B-type natriuretic peptide (NT-proBNP) validated for diagnosis of undifferentiated acutely decompensated heart failure (ADHF) remain useful in patients with heart failure with preserved ejection fraction (HFPEF), with minor loss of diagnostic performance.

 

  • BNP and NT-proBNP measured on admission with ADHF are powerfully predictive of in-hospital mortality in both HFPEF and heart failure with reduced EF (HFREF), with similar or greater risk in HFPEF as in HFREF associated with any given level of either peptide.

 

  • In stable treated heart failure, plasma natriuretic peptide concentrations often fall below cut-point values used for the diagnosis of ADHF in the emergency department; in HFPEF, levels average approximately half those in HFREF.

 

  • BNP and NT-proBNP are powerful independent prognostic markers in both chronic HFREF and chronic HFPEF, and the risk of important clinical adverse outcomes for a given peptide level is similar regardless of left ventricular ejection fraction.

 

  • Serial measurement of BNP or NT-proBNP to monitor status and guide treatment in chronic heart failure may be more applicable in HFREF than in HFPEF.

 

In addition, they point out the following:

 

BNP and NT-proBNP fall below ADHF thresholds in stable HFREF in approximately 50% and 20% of cases, respectively. Levels in stable HFPEF are even lower, approximately half those in HFREF.

 

Whereas BNPs have 90% sensitivity for asymptomatic LVEF of less than 40% in the community (a precursor state for HFREF), they offer no clear guide to the presence of early community based HFPEF.

 

Guidelines recommend BNP and NT-proBNP as adjuncts to the diagnosis of acute and chronic HF and for risk stratification. Refinements for application to HFPEF are needed.

 

The prognostic power of NPs is similar in HFREF and HFPEF. Defined levels of BNP and NT-proBNP correlate with similar short-term and long-term risks of important clinical adverse outcomes in both HFREF and HFPEF.

 

They provide a diagnostic algorithm for suspected heart failure (75)(Figure 9).

 

Figure 9

Diagnostic algorithm for suspected heart failure presenting either acutely or nonacutely

 

 

Diagnostic algorithm for suspected heart failure presenting either acutely or nonacutely. a In the acute setting, mid-regional pro–atrial natriuretic peptide may also be used (cutoff point 120 pmol/L; ie, <120 pmol/L 5 heart failure unlikely). b Other causes of elevated natriuretic peptide levels in the acute setting are an acute coronary syndrome, atrial or ventricular arrhythmias, pulmonary embolism, and severe chronic obstructive pulmonary disease with elevated right heart pressures, renal failure, and sepsis. Other causes of an elevated natriuretic level in the nonacute setting are old age (>75 years), atrial arrhythmias, left ventricular hypertrophy, chronic obstructive pulmonary disease, and chronic kidney disease. c Exclusion cutoff points for natriuretic peptides are chosen to minimize the false-negative rate while reducing unnecessary referrals for echocardiography. Treatment may reduce natriuretic peptide concentration, and natriuretic peptide concentrations may not be markedly elevated in patients with heart failure with preserved ejection fraction.

 

Patients with acute pulmonary symptoms and with acute myocardial infarct present with dyspnea to the Emergency Department.  The evaluation is made particularly difficult in a patient for whom there is no prior history. Maisel et al. (76) presented the utility of the midregion proadrenomedullin (MR-proADM) in all patients presenting with acute shortness of breath.  They found that MR-proADM was superior to BNP or troponin for predicting 90-day all-cause mortality in patients presenting with acute dyspnea (c index = 0.755, p < 0.0001). Furthermore, MR-proADM added significantly to all clinical variables (all adjusted hazard ratios: HR=3.28), and it was also superior to all other biomarkers.

 

There is a large body of recent work that has enlarged our view of hypertension, kidney disease, cardiovascular disease, including heart failure with (HFpEF) or without preserved ejection fraction. I shall here refer to my review in Leaders in Pharmaceutical Innovation  (78).  The piece contains a study that I published  (79) with collaborators in Brooklyn, Bridgeport and Philadelphia that is no longer available from the publisher.

 

The natriuretic peptides, B-type natriuretic peptide (BNP) and NT-proBNP that have emerged as tools for diagnosing congestive heart failure (CHF) are affected by age and renal insufficiency (RI).  NTproBNP is used in rejecting CHF and as a marker of risk for patients with acute coronary syndromes. This observational study was undertaken to evaluate the reference value for interpreting NT-proBNP concentrations. The hypothesis is that increasing concentrations of NT-proBNP are associated with the effects of multiple co-morbidities, not merely CHF,

resulting in altered volume status or myocardial filling pressures.

 

NT-proBNP was measured in a population with normal trans-thoracic echocardiograms
(TTE) and free of anemia or renal impairment. Exclusion conditions were the following
co-morbidities:

 

 

  • anemia as defined by WHO,
  • atrial fibrillation (AF),
  • elevated troponin T exceeding 0.070 mg/dl,
  • systolic or diastolic blood pressure exceeding 140 and 90 respectively,
  • ejection fraction less than 45%,
  • left ventricular hypertrophy (LVH),
  • left ventricular wall relaxation impairment, and
  • renal insufficiency (RI) defined by creatinine clearance < 60ml/min using
    the MDRD formula .

Study participants were seen in acute care for symptoms of shortness of breath suspicious for CHF requiring evaluation with cardiac NTproBNP assay. The median NT-proBNP for patients under 50 years is 60.5 pg/ml with an upper limit of 462 pg/ml, and for patients over 50 years the median was 272.8 pg/ml with an upper limit of 998.2 pg/ml.

We suggested that NT-proBNP levels can be more accurately interpreted only after removal of the major co-morbidities that affect an increase in this  peptide in serum. The PRIDE study guidelines (http://www.pridestudy.org/)  should be applied until presence or absence of comorbidities is diagnosed. With no comorbidities, the reference range for normal over 50 years of age remains steady at ~1000 pg/ml. The effect shown in previous papers likely is due to increasing concurrent comorbidity with age.

We observed the following changes with respect to NTproBNP and age:

(i) Sharp increase in NT-proBNP at over age 50

(ii) Increase in NT-proBNP at 7% per decade over 50

(iii) Decrease in eGFR at 4% per decade over 50

(iv) Slope of NT-proBNP increase with age is related to proportion of patients with eGFR less than 90

(v) NT-proBNP increase can be delayed or accelerated based on disease comorbidities

The mean and 95% CI of NTproBNP (CHF removed) by the National Kidney Foundation staging for eGFR interval (eGFR scale: 0, > 120; 1, 90 to 119;2, 60 to 89; 3, 40 to 59; 4, 15 to 39; 5, under 15 ml/min). We created a new variable to minimize the effects of age and eGFR variability by correcting these large effects in the whole sample population.

Adjustment of the NT-proBNP for  both eGFR and for age over 50 differences. We have carried out a normalization to adjust for both eGFR and for age over 50:

(i) Take Log of NT-proBNP and multiply by 1000
(ii) Divide the result by eGFR (using MDRD9 or Cockroft Gault10)
(iii) Compare results for age under 50, 50-70, and over 70 years
(iv) Adjust to age under 50 years by multiplying by 0.66 and 0.56.

Figure 10

 

 

NKF staging by GFRe interval and NT-proBNP (CHF removed).

 

 

The equation does not require weight because the results are reported normalized

to 1.73 m2 body surface area, which is an accepted average adult surface area.

 

This is illustrated in Figure 11.

Figure 11

 

Plot of 1000*log (NT-proBNP)/GFR vs age at  eGFR over 90  and 60 ml/min

Figure 12 compares the reference ranges for NTproBNP before and after adjustment.

  • before adjustment; b) after adjustment. c) the scatterplot for 1000xlog(NT proBNP) versus 1000xlog(NT-proBNP/eGFR). Superimposed scatterplot and regression line with centroid and

confidence interval for 1000*log(NT-proBNP)/eGFR vs age (anemia removed)

at eGFR over 40 and 90 ml/min. (Black: eGFR > 90, Blue:  eGFR > 40)

 

More recent work is enlightening.  Hijazi et al. (80) studied the incremental value of measuring N-terminal pro–B-type natriuretic peptide (NT-proBNP) levels in addition to established risk factors (including the CHA2DS2VASc [heart failure, hypertension, age 75 years and older, diabetes, and previous stroke or transient ischemic attack, vascular disease, age 65 to 74 years, and sex category) for the prediction of cardiovascular and bleeding events. They concluded that NT-proBNP levels are often elevated in atrial fibrillation (AF) and it is independently associated with an increased risk for stroke and mortality. NT-proBNP improves risk stratification beyond the CHA2DS2VASc score and might be a novel tool for improved stroke prediction in AF. The

efficacy of apixaban compared with warfarin was independent of the NT-proBNP level. Moreover, natriuretic peptides are regulatory hormones associated with cardiac remodeling, namely, left ventricular hypertrophy and systolic/diastolic dysfunction. Another study reported that the risk of death of patients with plasma NT-proBNP 133 pg/mL (third tertile of the distribution) was 3.3 times that of patients with values 50.8 pg/mL (first tertile; hazard ratio: 3.30 [95% CI: 0.90 to 12.29]). This predictive value was independent of, and superior to, that of 2 ECG indexes of left ventricular hypertrophy, the Sokolov-Lyon index and the amplitude of the R wave in lead aVL and it persisted in patients without ECG left ventricular hypertrophy (81).
Many patients presenting with acute dyspnea (including those with ADHF) have multiple coexisting medical disorders that may complicate their diagnosis and management. These patients presenting with acute dyspnea may have longer hospital length of stay and are at high risk for repeat hospitalization or death. In this presentation testing for brain natriuretic peptide (BNP) or NT-proBNP has been shown to be valuable for an accurate and efficient diagnosis and prognostication of HF (82).

 

The biological activity of BNP, the product of an intracellular peptide (proBNP108) that is converted to NT-proBNP, includes stimulation of natriuresis and vasorelaxation; inhibition of renin, aldosterone, and sympathetic nervous activity; inhibition of fibrosis; and improvement in myocardial relaxation.

 

Figure 13

 

Biology of the natriuretic peptide system. BNP indicates brain natriuretic peptide; NT-proBNP, amino-terminal pro-B-type natriuretic peptide; and DPP-IV, dipeptidyl peptidase-4.

The authors remind us that approximately 20% of patients with acute dyspnea have BNP or NT-proBNP levels that are above the cutoff point to exclude HF but too low to definitively identify it (82). Knowledge of the differential diagnosis of non-HF elevation of NP, as well as interpretation of the BNP or NT-proBNP value in the context of a clinical assessment is essential.  Across all stages of HF, elevated BNP or NT-proBNP concentrations are at least comparable prognostic predictors of mortality and cardiovascular events relative to traditional predictors of outcome in this setting, with increasing NP concentrations predicting worse prognosis in a linear fashion. This prognostic value may be used to stratify patients at the highest risk of adverse outcomes (see Figure 2 In this page). Age-adjusted Kaplan-Meier survival curve of mortality at 1 year associated with an elevated amino-terminal pro-B-type natriuretic peptide    (NT-proBNP) concentration at emergency department presentation with dyspnea in those with acutely decompensated heart failure. Reproduced from Januzzi et al22. (82)

The importance of determining diastolic and systolic function and for measurement of pulmonary artery pressure by echocardiography is clear, as NT-proBNP levels may be increased with increase in pulmonary pressure as well as conditions that increase cardiac output. Although Hijazi et al. used the Cockcroft-Gault (CG) equation to determine the glomerular filtration rate (GFR) the CG equation may find higher eGFR in older individuals (80). In addition, elevated NT-proBNP independently predicts all-cause mortality and morbidity of patients with AF. A prominent disease with elevated NT-proBNP is a respiratory system disease, such as chronic obstructive pulmonary disease, pulmonary embolism, and interstitial lung disease, in which B-type natriuretic peptide levels are elevated in response to the pressure of the right side of the heart. The authors conclude that one should keep in mind that NT-proBNP alone may be inadequate.

NT-proBNP level is used for the detection of acute CHF and as a predictor of survival. However, a number of factors, including renal function, may affect the NT-proBNP levels. This study aims to provide a more precise way of interpreting NT-proBNP levels based on GFR, independent of age. This study includes 247 pts in whom CHF and known confounders of elevated NT-proBNP were excluded, to show the relationship of GFR in association with age. The effect of eGFR on NT-proBNP level was adjusted by dividing 1000 x log(NT-proBNP) by eGFR then further adjusting for age in order to determine a normalized NT-proBNP value. The normalized NT-proBNP levels were affected by eGFR independent of the age of the patient. A normalizing function based on eGFR eliminates the need for an age-based reference ranges for NT-proBNP (79).

The routine use of natiuretic peptides in severely dyspneic patients has recently been called into question. We hypothesized that the diagnostic utility of Amino Terminal pro Brain Natiuretic Peptide (NT-proBNP) is diminished in a complex elderly population (83)

We studied 502 consecutive patients in whom NT-proBNP values were obtained to evaluate severe dyspnea in the emergency department (84). The diagnostic utility of NT-proBNP for the diagnosis of congestive heart failure (CHF) was assessed utilizing several published guidelines, as well as the manufacturer’s suggested age dependent cut-off points. The area under the receiver operator curve (AUC) for NT-proBNP was 0.70. Using age-related cut points, the diagnostic accuracy of NT-proBNP for the diagnosis of CHF was below prior reports (70% vs. 83%). Age and estimated creatinine clearance correlated directly with NT-proBNP levels, while hematocrit correlated inversely. Both age > 50 years and to a lesser extent hematocrit < 30% affected the diagnostic accuracy of NT-proBNP, while renal function had no effect. In multivariate analysis, a prior history of CHF was the best predictor of current CHF, odds ratio (OR) = 45; CI: 23-88.

The diagnostic accuracy of NT-proBNP for the evaluation of CHF appears less robust in an elderly population with a high prevalence of prior CHF. Age and hematocrit levels, may adversely affect the diagnostic accuracy off NT-proBNP (85).

Obesity and hypertension.

Obesity is associated with an increased risk of hypertension. In the past 5 years there have been dramatic advances into the genetic and neurobiological mechanisms of obesity with the discovery of leptin and novel neuropeptide pathways regulating appetite and metabolism. In this brief review, we argue that these mounting advances into the neurobiology of obesity have and will continue to provide new insights into the regulation of arterial pressure in obesity. We focus our comments on the sympathetic, vascular, and renal mechanisms of leptin and melanocortin receptor agonists and on the regulation of arterial pressure in rodent models of genetic obesity. Three concepts are proposed (86).

First, the effect of obesity on blood pressure may depend critically on the genetic-neurobiological mechanisms underlying the obesity. Second, obesity is not consistently associated with increased blood pressure, at least in rodent models. Third, the blood pressure response to obesity may be critically influenced by modifying alleles in the genetic background.

Leptin plays an important role in regulation of body weight through regulation of food intake and sympathetically mediated thermogenesis. The hypothalamic melanocortin system, via activation of the melanocortin-4 receptor (MC4-R), decreases appetite and weight, but its effects on sympathetic nerve activity (SNA) are unknown. In addition, it is not known whether sympathoactivation to leptin is mediated by the melanocortin system.

The following study (87) tested the interactions between these systems in regulation of brown adipose tissue (BAT) and renal and lumbar SNA in anesthetized Sprague-Dawley rats. Intracerebroventricular administration of the MC4-R agonist MT-II (200 to 600 pmol) produced a dose-dependent sympathoexcitation affecting BAT and renal and lumbar beds. This response was completely blocked by the MC4-R antagonist SHU9119 (30 pmol ICV). Administration of leptin (1000 m g/kg IV) slowly increased BAT SNA (baseline, 4166 spikes/s; 6 hours, 196628 spikes/s; P50.001) and renal SNA (baseline, 116616 spikes/s; 6 hours, 169626 spikes/s; P50.014).

Intracerebroventricular administration of SHU9119 did not inhibit leptin-induced BAT sympathoexcitation (baseline, 3567 spikes/s; 6 hours, 158634 spikes/s; P50.71 versus leptin alone). However, renal sympathoexcitation to leptin was completely blocked by SHU9119 (baseline, 142617 spikes/s; 6 hours, 146625 spikes/s; P50.007 versus leptin alone). The study (87) demonstrates that the hypothalamic melanocortin system can act to increase sympathetic nerve traffic to thermogenic BAT and other tissues. Our data also suggest that leptin increases renal SNA through activation of hypothalamic melanocortin receptors. In contrast, sympathoactivation to thermogenic BAT by leptin appears to be independent of the melanocortin system.

Troponins

The introduction of the first generation troponins T and I was an important event leading to the declining use of creatine kinase isoenzyme MB because of the short half-life in the circulation of CKMB and the possibility of missing a late presenting ACS. The situation then would call for the measurement of lactate dehydrogenase isoenzyme 1 (H-type), which had a decline in use.  The troponins T and I are proteins associated with the muscle contractile element with high specificity for the cardiomyocyte apparatus, which increased rapidly after ACS and which had estimated diagnostic cutoffs of 0.08 mg/dl and 1 mg/dl respectively.  The choice of marker was largely dependent of the instrument platform.  These biomarkers went through several generations of improvement to improve the diagnostic sensitivity to a cutoff at 2 SD of the lower limit of detection, magnifying confusion in interpretation that had always existed. These cardiospecific markers are elevated in patients with hypertension and specifically, long term CKD. This was clarified by introducing the terms Type 1 and Type 2 myocardial infarct, designating the classic ACS due to plaque rupture as Type 1.  However, the type 2 class might well be non-homogeneous. In any case, these are the best we have in detecting myocardial ischemic damage with biomarker release.

 

Discussion

This discussion has covered a large body of research involving hypertension, the kidney, and cardiovascular humoral mechanisms of control with a broad brush.  The work that has been done is far more than is cited.  There are several biomarkers that we have considered. They are not only laboratory based measurements.  They are: PWV, cystatin C, eGFR, copeptin, BNP or NT-BNP, Midregional prohormone adrenomedullin (MR-ADM), urinary albumin excretion, and the aldosterone/renin ratio.

The preceding discussion reminds us of the story of the blind men palpating an elephant, set in a poem by John Godfrey Saxe. These blind men were asked to tell of their experiences palpating different parts of an elephant, without seeing the entire animal Figure 1. Each of the blind men was able to palpate one part of the elephant, and thus was able to describe it in terms that were “partly in the right.” However, because none of them was able to encompass the entire elephant in their hands, they were also “in the wrong,” in that they failed to identify the whole elephant (88).
The blind men and the elephant. Poem by John Godfrey Saxe (Cartoon originally copyrighted by the authors (88); G. Renee Guzlas, artist). http://www.nature.com/ki/journal/v62/n5/thumbs/4493262f1bth.gif

These authors advanced the “elephant” as the increased oxidative burden in the uremic milieu of patients with chronic kidney disease. I introduce the concept in the diagnostic dilemma about what biomarkers are diagnostically informative in hypertension and ischemic CVD poses a conundrum. In reviewing the full gamut of biomarkers, we have a replay of the Lone Ranger and the silver bullet.  The problem is that there is no “silver” bullet.  We are accustomed to rely on clinical observations that are themselves weak covariates in actual experience.  The studies that have been done to validate the effectiveness of key biomarkers are well designed and show relevance in the populations studied.  However, they are insufficient by themselves in the emergent care population.
 

Impediments to a solution to the problem

Tests are ordered by physicians based on the findings in a clinical history and physical examination. Test that are ordered are reimbursed by insurance carriers, Medicare and Medicaid based on a provisional diagnosis.  The provisional diagnosis generates an ICD10 code, which has been most recently revised with a weighted input from the insurers that is not in favor of considered clinical evidence.  Moreover, the provider of care is graded based on the number of patients seen and the tests performed on a daily basis over any period.  Given this situation, and in addition, the requirement to interact with an outmoded information system that is more helpful to the insurer and less helpful to the provider, it is not surprising that there is a large burnout of the nursing and physician practitioner workforce.  If the diagnosis is inconclusive at the time of patient examination, then the work is not reimbursable based on ICD10 coding requirements that are disease specific.   This problem breaks down into a workload and a reimbursement inconsistency, neither of which makes sense in terms of the original studies on Diagnosis Related Groups (89) at Yale by Robert Fetter’s group.  The problem is made worse by the design and selection of healthcare information systems.

Many have pointed out the flaws in current EHR design that impede the optimum use of data and hinder workflow. Researchers have suggested that EHRs can be part of a learning health system to better capture and use data to improve clinical practice, create new evidence, educate, and support research efforts. The health care system suffers from both inefficient and ineffective use of data. Data are suboptimally displayed to users, undernetworked, underutilized, and wasted. Errors, inefficiencies, and increased costs occur on the basis of unavailable data in a system that does not coordinate the exchange of information, or adequately support its use (90). Clinicians’ schedules are stretched to the limit and yet the system in which they work exerts little effort to streamline and support carefully engineered care processes. Information for decision-making is difficult to access in the context of hurried real-time workflows(91)

 

 

The solution to the problem

The current design of the Electronic Medical Record (EMR) is a linear presentation of portions of the record by services, by diagnostic method, and by date, to cite examples.  This allows perusal through a graphical user interface (GUI) that partitions the information or necessary reports in a workstation entered by keying to icons.  This requires that the medical practitioner finds the history, medications, laboratory reports, cardiac imaging and EKGs, and radiology in different workspaces.  The introduction of a DASHBOARD has allowed a presentation of drug reactions, allergies, primary and secondary diagnoses, and critical information about any patient the care giver needing access to the record.  The advantage of this innovation is obvious.  The startup problem is what information is presented and how it is displayed, which is a source of variability and a key to its success.

Gil David and Larry Bernstein have developed, in consultation with Prof. Ronald Coifman, in the Yale University Applied Mathematics Program, a software system that is the equivalent of an intelligent Electronic Health Records Dashboard (92)( that provides empirical medical reference and suggests quantitative diagnostics options.

The most commonly ordered test used for managing patients worldwide is the hemogram that often incorporates the review of a peripheral smear.  While the hemogram has undergone progressive modification of the measured features over time the subsequent expansion of the panel of tests has provided a window into the cellular changes in the production, release or suppression of the formed elements from the blood-forming organ to the circulation.  In the hemogram one can view data reflecting the characteristics of a broad spectrum of medical conditions.

How we frame our expectations is so important that it determines the data we collect to examine the process.   In the absence of data to support an assumed benefit, there is no proof of validity at whatever cost.   This has meaning for hospital operations, for nonhospital laboratory operations, for companies in the diagnostic business, and for planning of health systems.

In 1983, a vision for creating the EMR was introduced by Lawrence Weed, expressed by McGowan and Winstead-Fry (93)

The data presented has to be comprehended in context with vital signs, key symptoms, and an accurate medical history.  Consequently, the limits of memory and cognition are tested in medical practice on a daily basis.  We deal with problems in the interpretation of data presented to the physician, and how through better design of the software that presents this data the situation could be improved.  The computer architecture that the physician uses to view the results is more often than not presented as the designer would prefer, and not as the end-user would like.

Eugene Rypka contributed greatly to clarifying the extraction of features (94) in a series of articles, which set the groundwork for the methods used today in clinical microbiology.  The method he describes is termed S-clustering, and will have a significant bearing on how we can view hematology data.  He describes S-clustering as extracting features from endogenous data that amplify or maximize structural information to create distinctive classes.  The method classifies by taking the number of features with sufficient variety to map into a theoretic standard. The mapping is done by a truth table, and each variable is scaled to assign values for each: message choice.  The number of messages and the number of choices forms an N-by N table.  He points out that the message choice in an antibody titer would be converted from 0 + ++ +++ to 0 1 2 3.

Bernstein and colleagues had a series of studies using Kullback-Liebler Distance  (effective information) for clustering to examine the latent structure of the elements commonly used for diagnosis of myocardial infarction (95-97)(CK-MB, LD and the isoenzyme-1 of LD),  protein-energy malnutrition (serum albumin, serum transthyretin, condition associated with protein malnutrition (see Jeejeebhoy and subjective global assessment), prolonged period with no oral intake), prediction of respiratory distress syndrome of the newborn (RDS), and prediction of lymph nodal involvement of prostate cancer, among other studies.   The exploration of syndromic classification has made a substantial contribution to the diagnostic literature, but has only been made useful through publication on the web of calculators and nomograms (such as Epocrates and Medcalc) accessible to physicians through an iPhone.  These are not an integral part of the EMR, and the applications require an anticipation of the need for such processing.

Gil David et al. (90, 92) introduced an AUTOMATED processing of the data available to the ordering physician and can anticipate an enormous impact in diagnosis and treatment of perhaps half of the top 20 most common causes of hospital admission that carry a high cost and morbidity.  For example: anemias (iron deficiency, vitamin B12 and folate deficiency, and hemolytic anemia or myelodysplastic syndrome); pneumonia; systemic inflammatory response syndrome (SIRS) with or without bacteremia; multiple organ failure and hemodynamic shock; electrolyte/acid base balance disorders; acute and chronic liver disease; acute and chronic renal disease; diabetes mellitus; protein-energy malnutrition; acute respiratory distress of the newborn; acute coronary syndrome; congestive heart failure; disordered bone mineral metabolism; hemostatic disorders; leukemia and lymphoma; malabsorption syndromes; and cancer(s)[breast, prostate, colorectal, pancreas, stomach, liver, esophagus, thyroid, and parathyroid]. The same approach has also been applied to the problem of hospital malnutrition, but it has not been sufficiently applied to hypertension, cardiovascular diseases, acute coronary syndrome, chronic renal failure.

We have developed (David G, Bernstein L, and Coifman) (92) a software system that is the equivalent of an intelligent Electronic Health Records Dashboard that provides empirical medical reference and suggests quantitative diagnostics options. The primary purpose is to gather medical information, generate metrics, analyze them in realtime and provide a differential diagnosis, meeting the highest standard of accuracy. The system builds its unique characterization and provides a list of other patients that share this unique profile, therefore utilizing the vast aggregated knowledge (diagnosis, analysis, treatment, etc.) of the medical community. The main mathematical breakthroughs are provided by accurate patient profiling and inference methodologies in which anomalous subprofiles are extracted and compared to potentially relevant cases. As the model grows and its knowledge database is extended, the diagnostic and the prognostic become more accurate and precise. We anticipate that the effect of implementing this diagnostic amplifier would result in higher physician productivity at a time of great human resource limitations, safer prescribing practices, rapid identification of unusual patients, better assignment of patients to observation, inpatient beds, intensive care, or referral to clinic, shortened length of patients ICU and bed days.

The main benefit is a real time assessment as well as diagnostic options based on comparable cases, flags for risk and potential problems as illustrated in the following case acquired on 04/21/10. The patient was diagnosed by our system with severe SIRS at a grade of 0.61 .

Method for data organization and classification via characterization metrics.

The database is organized to enable linking a given profile to known profiles. This is achieved by associating a patient to a peer group of patients having an overall similar profile, where the similar profile is obtained through a randomized search for an appropriate weighting of variables. Given the selection of a patients’ peer group, we build a metric that measures the dissimilarity of the patient from its group. This is achieved through a local iterated statistical analysis in the peer group.

This characteristic metric is used to locate other patients with similar unique profiles, for each of whom we repeat the procedure described above. This leads to a network of patients with similar risk condition. Then, the classification of the patient is inferred from the medical known condition of some of the patients in the linked network.

How do we organize the data and linkages provided in the first place?

Predictors: PWV, cystatin C, creatinine, urea, eGFR, copeptin, BNP or NT-BNP, TnI or TnT, Midregional prohormone adrenomedullin (MR-ADM), urinary albumin excretion, and the aldosterone/renin ratio, homocysteine, transthyretin, glucose, albumin, chol/LDL, LD, Na+, K+,  Cl, HCO3, pH.

Conditions: AMI, CRF, ARF, hypertension, HFpEF, HFcEF, ADHF, obesity, PHT, RVHF, pulmonary edema, PEM

Other variables: sex (M,F), age, BMI. …

Conditioning data: take log transform for large ascending values, OR take deciles of variables, if necessary.  This could apply to NT-proBNP, BNP, TnI, TnT, CK and LD.

Arrange predictor variables in columns and patient-sequence in rows.  This is a bidimentional table.  The problem is to assign diagnoses to each patient-in sequence. There can be more than one diagnosis.

In reality the patient-sequence or identifier is not relevant. Only the condition assignment is.  The condition assignments are made in a column adjacent to the patient, and they fall into rows.
The construct appears to be a 2×2, but it is actually an n-dimensional  matrix.  Each patient position has one or more diagnoses.

Multivariate statistical analysis is used to extend this analysis to two or more predictors.   In this case a multiple linear regression or a linear discriminant function would be used to predict a dependent variable from two or more independent variables.   If there is linear association dependency of the variables is assumed and the test of hypotheses requires that the variances of the predictors are normally distributed.  A method using a log-linear model circumvents the problem of the distributional dependency in a method called ordinal regression.    There is also a relationship of analysis of variance, a method of examining differences between the means of  two or more groups.  Then there is linear discriminant analysis, a method by which we examine the linear separation between groups rather than the linear association between groups.  Finally, the neural network is a nonlinear, nonparametric model for classifying data with several variables into distinct classes. In this case we might imagine a curved line drawn around the groups to divide the classes. The focus of this discussion will be the use of linear regression  and explore other methods for classification purposes (98).

The real issue is how a combination of variables falls into a table with meaningful information.  We are concerned with accurate assignment into uniquely variable groups by information in test relationships. One determines the effectiveness of each variable by its contribution to information gain in the system.  The reference or null set is the class having no information.  Uncertainty in assigning to a classification is only relieved by providing sufficient information.  One determines the effectiveness of each variable by its contribution to information gain in the system.  The possibility for realizing a good model for approximating the effects of factors supported by data used for inference owes much to the discovery of Kullback-Liebler distance or “information” (99), and Akaike (100) found a simple relationship between K-L information and Fisher’s maximized log-likelihood function. A solid foundation in this work was elaborated by Eugene Rypka (101).  Of course, this was made far less complicated by the genetic complement that defines its function, which made more accessible the study of biochemical pathways.  In addition, the genetic relationships in plant genetics were accessible to Ronald Fisher for the application of the linear discriminant function.    In the last 60 years the application of entropy comparable to the entropy of physics, information, noise, and signal processing, has been fully developed by Shannon, Kullback, and others,  and has been integrated with modern statistics, as a result of the seminal work of Akaike, Leo Goodman, Magidson and Vermunt, and unrelated work by Coifman. Dr. Magidson writes about Latent Class Model evolution:

The recent increase in interest in latent class models is due to the development of extended algorithms which allow today’s computers to perform LC analyses on data containing more than just a few variables, and the recent realization that the use of such models can yield powerful improvements over traditional approaches to segmentation, as well as to cluster, factor, regression and other kinds of analysis.

Perhaps the application to medical diagnostics had been slowed by limitations of data capture and computer architecture as well as lack of clarity in definition of what are the most distinguishing features needed for diagnostic clarification.  Bernstein and colleagues (102-104) had a series of studies using Kullback-Liebler Distance  (effective information) for clustering to examine the latent structure of the elements commonly used for diagnosis of myocardial infarction (CK-MB, LD and the isoenzyme-1 of LD),  protein-energy malnutrition (serum albumin, serum transthyretin, condition associated with protein malnutrition (see Jeejeebhoy and subjective global assessment), prolonged period with no oral intake), prediction of respiratory distress syndrome of the newborn (RDS), and prediction of lymph nodal involvement of prostate cancer, among other studies.   The exploration of syndromic classification has made a substantial contribution to the diagnostic literature, but has only been made useful through publication on the web of calculators and nomograms (such as Epocrates and Medcalc) accessible to physicians through an iPhone.  These are not an integral part of the EMR, and the applications require an anticipation of the need for such processing.

Gil David et al. introduced an AUTOMATED processing of the data (104) available to the ordering physician and can anticipate an enormous impact in diagnosis and treatment of perhaps half of the top 20 most common causes of hospital admission that carry a high cost and morbidity.  For example: anemias (iron deficiency, vitamin B12 and folate deficiency, and hemolytic anemia or myelodysplastic syndrome); pneumonia; systemic inflammatory response syndrome (SIRS) with or without bacteremia; multiple organ failure and hemodynamic shock; electrolyte/acid base balance disorders; acute and chronic liver disease; acute and chronic renal disease; diabetes mellitus; protein-energy malnutrition; acute respiratory distress of the newborn; acute coronary syndrome; congestive heart failure; disordered bone mineral metabolism; hemostatic disorders; leukemia and lymphoma; malabsorption syndromes; and cancer(s)[breast, prostate, colorectal, pancreas, stomach, liver, esophagus, thyroid, and parathyroid].

Our database organized to enable linking a given profile to known profiles(102-104). This is achieved by associating a patient to a peer group of patients having an overall similar profile, where the similar profile is obtained through a randomized search for an appropriate weighting of variables. Given the selection of a patients’ peer group, we build a metric that measures the dissimilarity of the patient from its group. This is achieved through a local iterated statistical analysis in the peer group.

We then use this characteristic metric to locate other patients with similar unique profiles, for each of whom we repeat the procedure described above. This leads to a network of patients with similar risk condition. Then, the classification of the patient is inferred from the medical known condition of some of the patients in the linked network. Given a set of points (the database) and a newly arrived sample (point), we characterize the behavior of the newly arrived sample, according to the database. Then, we detect other points in the database that match this unique characterization. This collection of detected points defines the characteristic neighborhood of the newly arrived sample. We use the characteristic neighborhood in order to classify the newly arrived sample. This process of differential diagnosis is repeated for every newly arrived point.   The medical colossus we have today has become a system out of control and beset by the elephant in the room – an uncharted complexity.

 

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  12. Parag C. Patel, Colby R. Ayers, Sabina A. Murphy, et al. Association of Cystatin C with Left Ventricular Structure and Function (The Dallas Heart Study). Circulation: Heart Failure. 2009; 2: 98-104.  http://dx.doi.org:/10.1161/CIRCHEARTFAILURE.108.807271.
  13. Rule AD, Bergstralh EJ, Slezak JM, Bergert J, Larson TS. Glomerular filtraton rate estimated by cystatin C among different clinical presentations. Kidney Int. 2006; 69:399–405. http://dx.doi.org:/10.1038/sj.ki.5000073
  14. Muller B, Morgenthaler N, Stolz D, et al. Circulating levels of copeptin, a novel biomarker, in lower respiratory tract infections. Eur J Clin Invest 2007;37, 145–152.
  15. Stoiser B, Mortl D, Hulsmann M, et al. Copeptin, a fragment of the vasopressin precursor, as a novel predictor of outcome in heart failure.  Eur J Clin Invest Nov 2006; 36(11):771–778.
    http://dx.doi.org:/10.1111/j.1365-2362.2006.01724.x
  16. Meijer E, Bakker SJL, Helbesma N, et al. Copeptin, a surrogate marker of vasopressin, is associated with microalbuminuria in a large population cohort.  Kidney Intl 2010; 77:29–36.
    http://dx.doi.org:/10.1038/ki.2009.397
  17. Nickel NP, Lichtinghagen R, Golpon H, et al. Circulating levels of copeptin predict outcome in patients with pulmonary arterial hypertension. Respir Res. Nov 19, 2013; 14:130. http://dx.doi.org:/10.1186/1465-9921-14-130
  18. Tenderenda-Banasiuk E,  Wasilewska A, Filonowicz R, et al. Serum copeptin levels in adolescents with primary hypertension. Pediatr Nephrol. 2014; 29(3): 423–429.    doi:  10.1007/s00467-013-2683-5
  19. Richards M, Januzzi JL, and Troughton RW. Natriuretic Peptides in Heart Failure with Preserved Ejection Fraction.  Heart Failure Clin 2014; 10:453–470. http://dx.doi.org/10.1016/j.hfc.2014.04.006
  20. Maisel A, Mueller C, Nowak M and Peacock WF, et al. Midregion Prohormone Adrenomedullin and Prognosis in Patients Presenting with Acute Dyspnea Results from the BACH (Biomarkers in Acute Heart Failure) Trial. J Am Coll Cardiol 2011; 58(10):1057–67.  http://dx.doi.org:/10.1016/j.jacc.2011.06.006.
  21. Bernstein LH. Heart-Lung-Kidney: Essential Ties. Leaders in Pharmaceutical Innovation. http://pharmaceuticalinnovations.com
  22. Bernstein LH, Zions MY, Alam ME, et al.  What is the best approximation of reference normal for NT-proBNP? Clinical levels for enhanced assessment of NT-proBNP (CLEAN). J Med Lab and Diag 04/2011; 2:16-21. http://www.academicjournals.org/jmld
  23. Hijazi  Z., Wallentin  L., Siegbahn  A., et al; N-terminal pro-B-type natriuretic peptide for risk assessment in patients with atrial fibrillation: insights from the ARISTOTLE trial (Apixaban for the Prevention of Stroke in Subjects With Atrial Fibrillation. J Am Coll Cardiol. 2013; 61:2274-2284
  24. Paget V, Legedz L, Gaudebout N, et al. N-Terminal Pro-Brain Natriuretic Peptide A Powerful Predictor of Mortality in Hypertension. Hypertension. 2011; 57:702-709   http://hyper.ahajournals.org/content/57/4/702.full.pdf]
  25. Kim Han-Naand  Januzzi JL.  Natriuretic Peptide Testing in Heart Failure. Circulation 2011;  123: 2015-2019. http://dx.doi.org:/10.1161/CIRCULATIONAHA.110.979500
  26. Balta S, Demirkol S, Aydogan M, and Celik T. Higher N-Terminal Pro–B-Type Natriuretic Peptide May Be Related to Very Different Conditions.  J Am Coll Cardiol. 2013; 62(17):1634-1635.   http://dx.doi.org:/10.1016/j.jacc.2013.04.093
  27. Bernstein LH1, Zions MY, Haq SA, et al. Effect of renal function loss on NT-proBNP level variations. Clin Biochem. 2009 Jul; 42(10-11): 1091-8. http://dx.doi.org:/10.1016/j.clinbiochem.2009.02.027
  28. Afaq MA, Shoraki A, Oleg I, Bernstein L, and Stuart W. Zarich.  Validity of Amino Terminal pro-Brain Natiuretic Peptide in a Medically Complex Elderly Population. J Clin Med Res. 2011 Aug; 3(4): 156–163.   doi:  10.4021/jocmr606w
  29. Mark AL, Correia M, MorganDA, et al. New Concepts From the Emerging Biology of Obesity. Hypertension. 1999; 33[part II]:537-541.
  30. Himmelfarb J, Stenvinkel P, Ikizler TA and Hakim RM. The elephant in uremia: Oxidant stress as a unifying concept of cardiovascular disease in uremia. Kidney International (2002) 62, 1524–1538; http://dx.doi.org:/10.1046/j.1523-1755.2002.00600.x  http://www.nature.com/ki/journal/v62/n5/full/4493262a.html
  31. The blind men and the elephant. Poem by John Godfrey Saxe (Cartoon originally copyrighted by the authors; G. Renee Guzlas, artist). http://www.nature.com/ki/journal/v62/n5/thumbs/4493262f1bth.gif
  32. Fetter RB. Diagnosis Related Groups: Understanding Hospital Performance. Interfaces Jan. – Feb., 1991; 21(1), Franz Edelman Award Papers: 6-26
  33. Bernstein LH. Inadequacy of EHRs. Pharmaceutical Intelligence. https://pharmaceuticalintelligence.com/2015/11/05/inadequacy-of-ehrs/
  34. Celi LA,  Marshall JD, Lai Y, Stone DJ. Disrupting Electronic Health Records Systems: The Next Generation.  JMIR  Med Inform 2015 (23.10.15);  3(4) :e34
    http://dx.doi.org:/10.2196/medinform.4192
  35. Realtime Clinical Expert Support. Pharmaceutical Intelligence.  https://pharmaceuticalintelligence.com/2015/05/10/realtime-clinical-expert-support/
  36. McGowan JJ and Winstead-Fry P. Problem Knowledge Couplers: reengineering evidence-based medicine through interdisciplinary development, decision support, and research. Bull Med Libr Assoc. 1999 October;  87(4):462–470.)
  37. Rypka EW and Babb R. Automatic construction and use of an identification scheme. In MEDICAL RESEARCH ENGINEERING Apr 19709; (2):9-19. https://www.researchgate.net/publication/17720773_Automatic_construction_and_use_of_an_identification_scheme
  38. Rudolph, R. A., Bernstein, L. H. and Babb, J. Information induction for predicting acute myocardial infarction. Clinical Chemistry 1988; 34: 2031-2038.
  39. Bernstein LH, Qamar A, McPherson C, Zarich S. Evaluating a new graphical ordinal logit method (GOLDminer) in the diagnosis of myocardial infarction utilizing clinical features and laboratory data. Yale J Biol Med 1999; 72:259-268.
  40. Bernstein LH, Good IJ, Holtzman, Deaton ML, Babb J. Diagnosis of acute myocardial infarction from two measurements of creatine kinase isoenzyme MB with use of nonparametric probability estimation. Clin Chem 1989; 35(3):444-447.
  41. Bernstein LH. Regression: A richly textured method for comparison and classification of predictor variables. https://pharmaceuticalintelligence.com/2012/08/14/regression-a-richly-textured-method-for-comparison-and-classification-of-predictor-variables/
  42. Posada D and Buckley TR. Model Selection and Model Averaging in Phylogenetics: Advantages of Akaike Information Criterion and Bayesian Approaches over Likelihood Ratio Tests. Syst. Biol. 200; 53(5):793–808. http://dx.doi.org:/10.1080/10635150490522304
  1. Kullback S. and Leibler R. On Information and Sufficiency. Ann Math Statistics. Mar 1951; 22(1):79-86. http://www.csee.wvu.edu/~xinl/library/papers/math/statistics/Kullback_Leibler_1951.pdf
  2. Bernstein LH, David G, Rucinski J, Coifman RR. Converting Hematology Based Data Into an Inferential Interpretation. In INTECH Open Access Publisher, 2012. https://books.google.com/books/about/Converting_Hematology_Based_Data_Into_an.html
  3. Bernstein LH, David G, Coifman RR. Generating Evidence Based Interpretation of Hematology Screens via Anomaly Characterization. Open Clin Chem J 2011; 4:10-16
  4. Bernstein LH. Automated Inferential Diagnosis of SIRS, sepsis, septic shock. Medical Informatics View. https://pharmaceuticalintelligence.com/2012/08/01/automated-inferential-diagnosis-of-sirs-sepsis-septic-shock/
  5. Bernstein LH, David G, Coifman RR. The Automated Nutritional Assessment. Nutrition  2013; 29: 113-121

 

Other related articles published in this Open Access Online Scientific Journal include the following: 

Biomarkers and risk factors for cardiovascular events, endothelial dysfunction, and thromboembolic complications

Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H Bernstein, MD, FCAP

Summary – Volume 4, Part 2: Translational Medicine in Cardiovascular Diseases

Pathophysiological Effects of Diabetes on Ischemic-Cardiovascular Disease and on Chronic Obstructive Pulmonary Disease (COPD)

Assessing Cardiovascular Disease with Biomarkers

Endothelial Function and Cardiovascular Disease

Adenosine Receptor Agonist Increases Plasma Homocysteine

Inadequacy of EHRs

Innervation of Heart and Heart Rate

Biomarker Guided Therapy

Pharmacogenomics

The Union of Biomarkers and Drug Development

Natriuretic Peptides in Evaluating Dyspnea and Congestive Heart Failure

Epilogue: Volume 4 – Translational, Post-Translational and Regenerative Medicine in Cardiology

Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker

Biomarkers: Diagnosis and Management, Present and Future

Genetic Analysis of Atrial Fibrillation

Landscape of Cardiac Biomarkers for Improved Clinical Utilization

Fractional Flow Reserve (FFR) & Instantaneous wave-free ratio (iFR): An Evaluation of Catheterization Lab Tools for Ischemic Assessment

Dealing with the Use of the High Sensitivity Troponin (hs cTn) Assays

Cardiotoxicity and Cardiomyopathy Related to Drugs Adverse Effects

Amyloidosis with Cardiomyopathy

Accurate Identification and Treatment of Emergent Cardiac Events

The potential contribution of informatics to healthcare is more than currently estimated

Realtime Clinical Expert Support

Metabolomics is about Metabolic Systems Integration

Automated Inferential Diagnosis of SIRS, sepsis, septic shock

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Kurzweill Reports in Medical Science I

Curator: Larry H. Bernstein, MD, FCAP

 

 

 

E-coli bacteria found in some China farms and patients cannot be killed with antiobiotic drug of last resort

“One of the most serious global threats to human health in the 21st century” — could spread around the world, requiring “urgent coordinated global action”
November 20, 2015

http://www.kurzweilai.net/e-coli-bacteria-found-in-some-china-farms-and-patients-cannot-be-killed-with-antiobiotic-drug-of-last-resort

Colistin antibiotic overused in farm animals in China apparently caused E-coli bacteria to become completely resistant to treatment; E-coli strain has already spread to Laos and Malaysia (credit: Yi-Yun Liu et al./Lancet Infect Dis)

Widespread E-coli bacteria that cannot be killed with the antiobiotic drug of last resort — colistin — have been found in samples taken from farm pigs, meat products, and a small number of patients in south China, including bacterial strains with epidemic potential, an international team of scientists revealed in a paper published Thursday Nov. 19 in the journal The Lancet Infectious Diseases.

The scientists in China, England, and the U.S. found a new gene, MCR-1, carried in E-coli bacteria strain SHP45. MCR-1 enables bacteria to be highly resistant to colistin and other polymyxins drugs.

“The emergence of the MCR-1 gene in China heralds a disturbing breach of the last group of antibiotics — polymixins — and an end to our last line of defense against infection,” said Professor Timothy Walsh, from the Cardiff University School of Medicine, who collaborated on this research with scientists from South China Agricultural University.

Walsh, an expert in antibiotic resistance, is best known for his discovery in 2011 of the NDM-1 disease-causing antibiotic-resistant superbug in New Delhi’s drinking water supply. “The rapid spread of similar antibiotic-resistant genes such as NDM-1 suggests that all antibiotics will soon be futile in the face of previously treatable gram-negative bacterial infections such as E.coli and salmonella,” he said.

Likely to spread worldwide; already found in Laos and Malaysia

The MCR-1 gene was found on plasmids — mobile DNA that can be easily copied and transferred between different bacteria, suggesting an alarming potential to spread and diversify between different bacterial populations.

Structure of plasmid pHNSHP45 carrying MCR-1 from Escherichia coli strain SHP45 (credit: Yi-Yun Liu et al./Lancet Infect Dis)

“We now have evidence to suggest that MCR-1-positive E.coli has spread beyond China, to Laos and Malaysia, which is deeply concerning,” said Walsh.  “The potential for MCR-1 to become a global issue will depend on the continued use of polymixin antibiotics, such as colistin, on animals, both in and outside China; the ability of MCR-1 to spread through human strains of E.coli; and the movement of people across China’s borders.”

“MCR-1 is likely to spread to the rest of the world at an alarming rate unless we take a globally coordinated approach to combat it. In the absence of new antibiotics against resistant gram-negative pathogens, the effect on human health posed by this new gene cannot be underestimated.”

“Of the top ten largest producers of colistin for veterinary use, one is Indian, one is Danish, and eight are Chinese,” The Lancet Infectious Diseases notes. “Asia (including China) makes up 73·1% of colistin production with 28·7% for export including to Europe.29 In 2015, the European Union and North America imported 480 tonnes and 700 tonnes, respectively, of colistin from China.”

Urgent need for coordinated global action

“Our findings highlight the urgent need for coordinated global action in the fight against extensively resistant and pan-resistant gram-negative bacteria,” the journal paper concludes.

“The implications of this finding are enormous,” an associated editorial comment to the The Lancet Infectious Diseases paper stated. “We must all reiterate these appeals and take them to the highest levels of government or face increasing numbers of patients for whom we will need to say, ‘Sorry, there is nothing I can do to cure your infection.’”

Margaret Chan, MD, Director-General of the World Health Organization, warned in 2011 that “the world is heading towards a post-antibiotic era, in which many common infections will no longer have a cure and, once again, kill unabated.”

“Although in its 2012 World Health Organization Advisory Group on Integrated Surveillance of Antimicrobial Resistance (AGISAR) report the WHO concluded that colistin should be listed under those antibiotics of critical importance, it is regrettable that in the 2014 Global Report on Surveillance, the WHO did not to list any colistin-resistant bacteria as part of their ‘selected bacteria of international concern,’” The Lancet Infectious Diseases paper says, reflecting WHO’s inaction in Ebola-stricken African countries, as noted last September by the international medical humanitarian organization Médecins Sans Frontières.

Funding for the E-coli bacteria study was provided by the Ministry of Science and Technology of China and National Natural Science Foundation of China.


Abstract of Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study

Until now, polymyxin resistance has involved chromosomal mutations but has never been reported via
horizontal gene transfer. During a routine surveillance project on antimicrobial resistance in commensal Escherichia coli from food animals in China, a major increase of colistin resistance was observed. When an E coli strain, SHP45, possessing colistin resistance that could be transferred to another strain, was isolated from a pig, we conducted further analysis of possible plasmid-mediated polymyxin resistance. Herein, we report the emergence of the first plasmid-mediated polymyxin resistance mechanism, MCR-1, in Enterobacteriaceae.

The mcr-1 gene in E coli strain SHP45 was identified by whole plasmid sequencing and subcloning. MCR-1 mechanistic studies were done with sequence comparisons, homology modelling, and electrospray ionisation mass spectrometry. The prevalence of mcr-1 was investigated in E coli and Klebsiella pneumoniae strains collected from five provinces between April, 2011, and November, 2014. The ability of MCR-1 to confer polymyxin resistance in vivo was examined in a murine thigh model.

Polymyxin resistance was shown to be singularly due to the plasmid-mediated mcr-1 gene. The plasmid carrying mcr-1 was mobilised to an E coli recipient at a frequency of 10−1 to 10−3 cells per recipient cell by conjugation, and maintained in K pneumoniae and Pseudomonas aeruginosa. In an in-vivo model, production of MCR-1 negated the efficacy of colistin. MCR-1 is a member of the phosphoethanolamine transferase enzyme family, with expression in E coli resulting in the addition of phosphoethanolamine to lipid A. We observed mcr-1 carriage in E coli isolates collected from 78 (15%) of 523 samples of raw meat and 166 (21%) of 804 animals during 2011–14, and 16 (1%) of 1322 samples from inpatients with infection.

The emergence of MCR-1 heralds the breach of the last group of antibiotics, polymyxins, by plasmid-mediated resistance. Although currently confined to China, MCR-1 is likely to emulate other global resistance mechanisms such as NDM-1. Our findings emphasise the urgent need for coordinated global action in the fight against pan-drug-resistant Gram-negative bacteria.

 

Researchers discover signaling molecule that helps neurons find their way in the developing brain

November 20, 2015

http://www.kurzweilai.net/researchers-discover-signaling-molecule-that-helps-neurons-find-their-way-in-the-developing-brain

This image shows a section of the spinal cord of a mouse embryo. Neurons appear green. Commissural axons (which connect the two sides of the brain) appear as long, u-shaped threads, and the bottom, yellow segment of the structure represents the midline (between brain hemispheres). (credit: Laboratory of Brain Development and Repair/ The Rockefeller University)

Rockefeller University researchers have discovered a molecule secreted by cells in the spinal cord that helps guide axons (neuron extensions) during a critical stage of central nervous system development in the embryo. The finding helps solve the mystery: how do the billions of neurons in the embryo nimbly reposition themselves within the brain and spinal cord, and connect branches to form neural circuits?

Working in mice, the researchers identified an axon guidance factor, NELL2, and explained how it makes commissural axons (which connect the two sides of the brain).

The findings could help scientists understand what goes wrong in a rare disease called horizontal gaze palsy with progressive scoliosis. People affected by the condition often suffer from abnormal spine curvature, and are unable to move their eyes horizontally from side to side. The study was published Thursday Nov. 19 in the journal Science.


Abstract of Operational redundancy in axon guidance through the multifunctional receptor Robo3 and its ligand NELL2

Axon pathfinding is orchestrated by numerous guidance cues, including Slits and their Robo receptors, but it remains unclear how information from multiple cues is integrated or filtered. Robo3, a Robo family member, allows commissural axons to reach and cross the spinal cord midline by antagonizing Robo1/2–mediated repulsion from midline-expressed Slits and potentiating deleted in colorectal cancer (DCC)–mediated midline attraction to Netrin-1, but without binding either Slits or Netrins. We identified a secreted Robo3 ligand, neural epidermal growth factor-like-like 2 (NELL2), which repels mouse commissural axons through Robo3 and helps steer them to the midline. These findings identify NELL2 as an axon guidance cue and establish Robo3 as a multifunctional regulator of pathfinding that simultaneously mediates NELL2 repulsion, inhibits Slit repulsion, and facilitates Netrin attraction to achieve a common guidance purpose.

A sensory illusion that makes yeast cells self-destruct

A possible tactic for cancer therapeutics
November 20, 2015

http://www.kurzweilai.net/a-sensory-illusion-that-makes-yeast-cells-self-destruct

 

Effects of osmotic changes on yeast cell growth. (A) Schematic of the flow chamber used to create osmotic level oscillations for different periods of time. (B) Cell growth for these periods. The graphs show the average number of progeny cells (blue) before and after applying stress for different periods (gray shows orginal “no stress” line). The inset shows representative images of cells for two periods. (credit: Amir Mitchell et al./Science)

UC San Francisco researchers have discovered that even brainless single-celled yeast have “sensory biases” that can be hacked by a carefully engineered illusion — a finding that could be used to develop new approaches to fighting diseases such as cancer.

In the new study, published online Thursday November 19 in Science Express, Wendell Lim, PhD, the study’s senior author*, and his team discovered that yeast cells falsely perceive a pattern of osmotic levels (by applying potassium chloride) that alternate in eight minute intervals as massive, continuously increasing stress. In response, the microbes over-respond and kill themselves. (In their natural environment, salt stress normally gradually increases.)

The results, Lim says, suggest a whole new way of looking at the perceptual abilities of simple cells and this power of illusion could even be used to develop new approaches to fighting cancer and other diseases.

“Our results may also be relevant for cellular signaling in disease, as mutations affecting cellular signaling are common in cancer, autoimmune disease, and diabetes,” the researchers conclude in the paper. “These mutations may rewire the native network, and thus could modify its activation and adaptation dynamics. Such network rewiring in disease may lead to changes that can be most clearly revealed by simulation with oscillatory inputs or other ‘non-natural’ patterns.

“The changes in network response behaviors could be exploited for diagnosis and functional profiling of disease cells, or potentially taken advantage of as an Achilles’ heel to selectively target cells bearing the diseased network.”

https://youtu.be/CuDjZrM8xtA
UC San Francisco (UCSF) | Sensory Illusion Causes Cells to Self-Destruct

* Chair of the Department of Cellular and Molecular Pharmacology at UCSF, director of the UCSF Center for Systems and Synthetic Biology, and a Howard Hughes Medical Institute (HHMI) investigator.

** Normally, sensor molecules in a yeast cell detect changes in salt concentration and instruct the cell to respond by producing a protective chemical. The researchers found that the cells were perfectly capable of adapting when they flipped the salt stress on and off every minute or every 32 minutes. But to their surprise, when they tried an eight-minute oscillation of precisely the same salt level the cells quickly stopped growing and began to die off.


Abstract of Oscillatory stress stimulation uncovers an Achilles’ heel of the yeast MAPK signaling network

Cells must interpret environmental information that often changes over time. We systematically monitored growth of yeast cells under various frequencies of oscillating osmotic stress. Growth was severely inhibited at a particular resonance frequency, at which cells show hyperactivated transcriptional stress responses. This behavior represents a sensory misperception—the cells incorrectly interpret oscillations as a staircase of ever-increasing osmolarity. The misperception results from the capacity of the osmolarity-sensing kinase network to retrigger with sequential osmotic stresses. Although this feature is critical for coping with natural challenges—like continually increasing osmolarity—it results in a tradeoff of fragility to non-natural oscillatory inputs that match the retriggering time. These findings demonstrate the value of non-natural dynamic perturbations in exposing hidden sensitivities of cellular regulatory networks.

Google Glass helps cardiologists complete difficult coronary artery blockage surgery

November 20, 2015

http://www.kurzweilai.net/google-glass-helps-cardiologists-in-challenging-coronary-artery-blockage-surgery

 

Google Glass allowed the surgeons to clearly visualize the distal coronary vessel and verify the direction of the guide wire advancement relative to the course of the occluded vessel segment. (credit: Maksymilian P. Opolski et al./Canadian Journal of Cardiology

Cardiologists from the Institute of Cardiology, Warsaw, Poland have used Google Glass in a challenging surgical procedure, successfully clearing a blockage in the right coronary artery of a 49-year-old male patient and restoring blood flow, reports the Canadian Journal of Cardiology.

Chronic total occlusion, a complete blockage of the coronary artery, sometimes referred to as the “final frontier in interventional cardiology,” represents a major challenge for catheter-based percutaneous coronary intervention (PCI), according to the cardiologists.

That’s because of the difficulty of recanalizing (forming new blood vessels through an obstruction) combined with poor visualization of the occluded coronary arteries.

Coronary computed tomography angiography (CTA) is increasingly used to provide physicians with guidance when performing PCI for this procedure. The 3-D CTA data can be projected on monitors, but this technique is expensive and technically difficult, the cardiologists say.

So a team of physicists from the Interdisciplinary Centre for Mathematical and Computational Modelling of theUniversity of Warsaw developed a way to use Google Glass to clearly visualize the distal coronary vessel and verify the direction of the guide-wire advancement relative to the course of the blocked vessel segment.

Three-dimensional reconstructions displayed on Google Glass revealed the exact trajectory of the distal right coronary artery (credit: Maksymilian P. Opolski et al./Canadian Journal of Cardiology)

The procedure was completed successfully, including implantation of two drug-eluting stents.

“This case demonstrates the novel application of wearable devices for display of CTA data sets in the catheterization laboratory that can be used for better planning and guidance of interventional procedures, and provides proof of concept that wearable devices can improve operator comfort and procedure efficiency in interventional cardiology,” said lead investigatorMaksymilian P. Opolski, MD, PhD, of the Department of Interventional Cardiology and Angiology at the Institute of Cardiology, Warsaw, Poland.

“We believe wearable computers have a great potential to optimize percutaneous revascularization, and thus favorably affect interventional cardiologists in their daily clinical activities,” he said. He also advised that “wearable devices might be potentially equipped with filter lenses that provide protection against X-radiation.


Abstract of First-in-Man Computed Tomography-Guided Percutaneous Revascularization of Coronary Chronic Total Occlusion Using a Wearable Computer: Proof of Concept

We report a case of successful computed tomography-guided percutaneous revascularization of a chronically occluded right coronary artery using a wearable, hands-free computer with a head-mounted display worn by interventional cardiologists in the catheterization laboratory. The projection of 3-dimensional computed tomographic reconstructions onto the screen of virtual reality glass allowed the operators to clearly visualize the distal coronary vessel, and verify the direction of the guide wire advancement relative to the course of the occluded vessel segment. This case provides proof of concept that wearable computers can improve operator comfort and procedure efficiency in interventional cardiology.

Modulating brain’s stress circuity might prevent Alzheimer’s disease

Drug significantly prevented onset of cognitive and cellular effects in mice
November 17, 2015

http://www.kurzweilai.net/modulating-brains-stress-circuity-might-prevent-alzheimers-disease

 

Effect of drug treatment on AD mice in control group (left) or drug (right) on Ab plaque load. (credit: Cheng Zhang et al./Alzheimer’s & Dementia: The Journal of the Alzheimer’s Association)

In a novel animal study design that mimicked human clinical trials, researchers at University of California, San Diego School of Medicine report that long-term treatment using a small-molecule drug that reduces activity of  the brain’s stress circuitry significantly reduces Alzheimer’s disease (AD) neuropathology and prevents onset of cognitive impairment in a mouse model of the neurodegenerative condition.

The findings are described in the current online issue of the journal Alzheimer’s & Dementia: The Journal of the Alzheimer’s Association.

Previous research has shown a link between the brain’s stress signaling pathways and AD. Specifically, the release of a stress-coping hormone called corticotropin-releasing factor (CRF), which is widely found in the brain and acts as a neurotransmitter/neuromodulator, is dysregulated in AD and is associated with impaired cognition and with detrimental changes in tau protein and increased production of amyloid-beta protein fragments that clump together and trigger the neurodegeneration characteristic of AD.

“Our work and that of our colleagues on stress and CRF have been mechanistically implicated in Alzheimer’s disease, but agents that impact CRF signaling have not been carefully tested for therapeutic efficacy or long-term safety in animal models,” said the study’s principal investigator and corresponding author Robert Rissman, PhD, assistant professor in the Department of Neurosciences and Biomarker Core Director for the Alzheimer’s Disease Cooperative Study (ADCS).

The researchers determined that modulating the mouse brain’s stress circuitry mitigated generation and accumulation of amyloid plaques widely attributed with causing neuronal damage and death. As a consequence, behavioral indicators of AD were prevented and cellular damage was reduced.  The mice began treatment at 30-days-old — before any pathological or cognitive signs of AD were present — and continued until six months of age.

One particular challenge, Rissman noted, is limiting exposure of the drug to the brain so that it does not impact the body’s ability to respond to stress. “This can be accomplished because one advantage of these types of small molecule drugs is that they readily cross the blood-brain barrier and actually prefer to act in the brain,” Rissman said.

“Rissman’s prior work demonstrated that CRF and its receptors are integrally involved in changes in another AD hallmark, tau phosphorylation,” said William Mobley, MD, PhD, chair of the Department of Neurosciences and interim co-director of the Alzheimer’s Disease Cooperative Study at UC San Diego. “This new study extends those original mechanistic findings to the amyloid pathway and preservation of cellular and synaptic connections.  Work like this is an excellent example of UC San Diego’s bench-to-bedside legacy, whereby we can quickly move our basic science findings into the clinic for testing,” said Mobley.

Rissman said R121919 was well-tolerated by AD mice (no significant adverse effects) and deemed safe, suggesting CRF-antagonism is a viable, disease-modifying therapy for AD. Drugs like R121919 were originally designed to treat generalized anxiety disorder, irritable bowel syndrome and other diseases, but failed to be effective in treating those disorders.

Rissman noted that repurposing R121919 for human use was likely not possible at this point. He and colleagues are collaborating with the Sanford Burnham Prebys Medical Discovery Institute to design new assays to discover the next generation of CRF receptor-1 antagonists for testing in early phase human safety trials.

“More work remains to be done, but this is the kind of basic research that is fundamental to ultimately finding a way to cure — or even prevent —Alzheimer’s disease,” said David Brenner, MD, vice chancellor, UC San Diego Health Sciences and dean of UC San Diego School of Medicine. “These findings by Dr. Rissman and his colleagues at UC San Diego and at collaborating institutions on the Mesa suggest we are on the cusp of creating truly effective therapies.”


Abstract of Corticotropin-releasing factor receptor-1 antagonism mitigates beta amyloid pathology and cognitive and synaptic deficits in a mouse model of Alzheimer’s disease

Introduction: Stress and corticotropin-releasing factor (CRF) have been implicated as mechanistically involved in Alzheimer’s disease (AD), but agents that impact CRF signaling have not been carefully tested for therapeutic efficacy or long-term safety in animal models.

Methods: To test whether antagonism of the type-1 corticotropin-releasing factor receptor (CRFR1) could be used as a disease-modifying treatment for AD, we used a preclinical prevention paradigm and treated 30-day-old AD transgenic mice with the small-molecule, CRFR1-selective antagonist, R121919, for 5 months, and examined AD pathologic and behavioral end points.

Results: R121919 significantly prevented the onset of cognitive impairment in female mice and reduced cellular and synaptic deficits and beta amyloid and C-terminal fragment-β levels in both genders. We observed no tolerability or toxicity issues in mice treated with R121919.

Discussion: CRFR1 antagonism presents a viable disease-modifying therapy for AD, recommending its advancement to early-phase human safety trials.

Allen Institute researchers decode patterns that make our brains human
Conserved gene patterning across human brains provide insights into health and disease
November 17, 2015

http://www.kurzweilai.net/allen-institute-researchers-decode-patterns-that-make-our-brains-human

 

Percentage of known neuron-, astrocyte- and oligodendrocyte-enriched genes in 32 modules, ordered by proportion of neuron-enriched gene membership. (credit: Michael Hawrylycz et al./Nature Neuroscience)

Allen Institute researchers have identified a surprisingly small set of just 32 gene-expression patterns for all 20,000 genes across 132 functionally distinct human brain regions, and these patterns appear to be common to all individuals.

In research published this month in Nature Neuroscience, the researchers used data for six brains from the publicly available Allen Human Brain Atlas. They believe the study is important because it could provide a baseline from which deviations in individuals may be measured and associated with diseases, and could also provide key insights into the core of the genetic code that makes our brains distinctly human.

While many of these patterns were similar in human and mouse, many genes showed different patterns in human. Surprisingly, genes associated with neurons were most conserved (consistent) across species, while those for the supporting glial cells showed larger differences. The most highly stable genes (the genes that were most consistent across all brains) include those associated with diseases and disorders like autism and Alzheimer’s, and these genes include many existing drug targets.

These patterns provide insights into what makes the human brain distinct and raise new opportunities to target therapeutics for treating disease.

The researchers also found that the pattern of gene expression in cerebral cortex is correlated with “functional connectivity” as revealed by neuroimaging data from the Human Connectome Project.

“The human brain is phenomenally complex, so it is quite surprising that a small number of patterns can explain most of the gene variability across the brain,” says Christof Koch, Ph.D., President and Chief Scientific Officer at the Allen Institute for Brain Science. “There could easily have been thousands of patterns, or none at all. This gives us an exciting way to look further at the functional activity that underlies the uniquely human brain.”


Abstract of Canonical genetic signatures of the adult human brain

The structure and function of the human brain are highly stereotyped, implying a conserved molecular program responsible for its development, cellular structure and function. We applied a correlation-based metric called differential stability to assess reproducibility of gene expression patterning across 132 structures in six individual brains, revealing mesoscale genetic organization. The genes with the highest differential stability are highly biologically relevant, with enrichment for brain-related annotations, disease associations, drug targets and literature citations. Using genes with high differential stability, we identified 32 anatomically diverse and reproducible gene expression signatures, which represent distinct cell types, intracellular components and/or associations with neurodevelopmental and neurodegenerative disorders. Genes in neuron-associated compared to non-neuronal networks showed higher preservation between human and mouse; however, many diversely patterned genes displayed marked shifts in regulation between species. Finally, highly consistent transcriptional architecture in neocortex is correlated with resting state functional connectivity, suggesting a link between conserved gene expression and functionally relevant circuitry.

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Leaders in Pharmaceutical Business Intelligence would like to announce their First Volume of their BioMedical E-Book Series A: eBooks on Cardiovascular Diseases

 

Perspectives on Nitric Oxide in Disease Mechanisms

Nitric Oxide coverwhich is now available on Amazon Kindle at

http://www.amazon.com/dp/B00DINFFYC

This book is a comprehensive review of Nitric Oxide, its discovery, function, and related opportunities for Targeted Therapy written by  Experts, Authors, Writers.  This book is a series of articles delineating the basic functioning of the NOS isoforms, their production widely by endothelial cells, and the effect of NITRIC OXIDE production by endothelial cells, by neutrophils and macrophages, the effect on intercellular adhesion, and the effect of circulatory shear and turbulence on NITRIC OXIDE production. The e-Book’s articles have been published on the  Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published, in real time in the e-Book which is a live book.

 

We invite e-Readers to write an Article Reviews on Amazon for this e-Book.

 

All forthcoming BioMed e-Book Titles can be viewed at:

https://pharmaceuticalintelligence.com/biomed-e-books/

 

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Perspectives on Nitric Oxide in Disease Mechanisms

Chapter 1: Nitric Oxide Basic Research

Chapter 2: Nitric Oxide and Circulatory Diseases

Chapter 3: Therapeutic Cardiovascular Targets

Chapter 4: Nitric Oxide and Neurodegenerative Diseases

Chapter 5: Bone Metabolism

Chapter 6: Nitric Oxide and Systemic Inflammatory Disease

Chapter 7: Nitric Oxide: Lung and Alveolar Gas Exchange

Chapter 8. Nitric Oxide and Kidney Dysfunction

Chapter 9: Nitric Oxide and Cancer 

 

 

 

 

 

 

 

 

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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle


Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD

 

Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

http://www.amazon.com/dp/B012BB0ZF0.

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

https://pharmaceuticalintelligence.com/biomed-e-books/

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 

 

Summary 

Epilogue

 

 

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Neutraceuticals

Author and Curator: Larry H Bernstein, MD, FCAP 

 

Introduction

This article is concerned with a very fast growing part of the food industry. Unfortunately, there have been such products that were sold that did not contain what was purported to provide the benefit. This segment of the medicinal industry is regulated as FOOD (food product), unlike pharmaceuticals. This means that the basic safety requirement is compliance with a labeling standard.  There are no clinical trials.  The incentive for the development in this sector is from 2,500 years of history in the use of plants for health benefits.  The new science that can improve the potency and the safety is the technology that has grown up to extract, refine and produce the active substance in pure form.  The methods used called Flow Chemistry has been covered recently by Anthony Melvin Crasso at his outstanding pharmaceutical site.  I shall list important articles that have been in the Pharmaceutical Intelligence site at the end of this discussion.

Antidiarrheal and antioxidant activities of chamomile (Matricaria recutita L.) decoction extract in rats

Hichem Sebai, Mohamed-Amine Jabri, Abdelaziz Souli, Kais Rtibi, et al.
J Ethnopharmacol 152(2014)327–332
http://dx.doi.org/10.1016/j.jep.2014.01.015

Ethnopharmacological relevance: Matricaria recutita L. (Chamomile) has been widely used in the Tunisian traditional medicine for the treatment of digestive system disorders. The present work aims to investigate the protective effects of chamomile decoction extract (CDE) against castor oil-induced diarrhea and oxidative stress in rats. Methods:The antidiarrheal activity was evaluated using castor oil-induced diarrhea method. In this respect, rats were divided into six groups: Control, Castor oil, Castor oil + Loperamide (LOP) and Castor oil + various doses of CDE. Animals were perorally (p.o.) pre-treated with CDE during 1 hand intoxicated for 2 or 4h by acute oral administration of castor oil.  Results: Our results showed that CDE produced a significant dose-dependent protection against castor oil-induced diarrhea and intestinal fluid accumulation. On the other hand, we showed that diarrhea was accompagnied by an oxidative stress status assessed by an increase of malondialdehyde (MDA) level and depletion of antioxidant enzyme activities as superoxide dismutase(SOD), catalase (CAT) and glutathione peroxidase (GPx). Castor oil also increased gastric and intestinal mucosa hydrogen peroxide (H2O2) and free iron levels. Importantly, we showed that chamomile pre-treatment abrogated all these biochemical alterations. Conclusion: These findings suggested that chamomile extract had a potent antidiarrheal and antioxidant properties in rats confirming their use in traditional medicine.

Anti-diarrheal activity of methanol extract of Santalum album L. in mice and gastrointestinal effect on the contraction of isolated jejunum in rats

Huimin Guo, Jingze Zhang, Wenyuan Gao, Zhuo Qu, Changxiao Liu
J Ethnopharmacol 54(2014)704–710
http://dx.doi.org/10.1016/j.jep.2014.04.043

Ethnopharmacological relevance: Santalum album L., namely Sandalwood, honoredas “Green Gold”, is a traditional Chinese herb which has the effects of antidiarrheal and antibacterial activity. But there is limited scientific study on its activity and mechanism in gastrointestinal disorders.
Materials and methods: in vivo, after intragastric administration, the methanol extract of Sandalwood (SE) (200, 400 and 800 mg/kg) were studied in castor oil-induced diarrhea mice. By the test of small intestinal hyperfunction induced by neostigmine, SE was studied on gastrointestinal transit including gastric emptying and small intestinal motility. Meanwhile, in vitro, the effects of SE (0.02, 0.05, 0.1, 0.2, 0.3, 0.4 mg/mL) on the isolated tissue preparations of rat jejunum were also investigated. The rat jejunum strips were precontacted with acetylcholine (Ach; 10-6M), 5-hydroxytryptamine (5-HT, 200 μM) or potassium chloride (KCl; 60mM) and tested in the presence of SE. In addition, the possible myogenic effect was analyzed in the pretreatment of the jejunum preparations with SE or verapamilin Ca 2+-free high-K+ (60mM) solution containing EDTA. Results: At doses of 200, 400 and 800 mg/kg, SE showed significant anti-diarrheal activity against castor oil-induced diarrhea as compared with the control. At the same doses, it also inhibited the gastric emptying and small intestinal motility in the mice of which small intestinal hyperfunction induced by neostigmine. It caused inhibitory effects on the spontaneous contraction of rat-isolated jejunum in dose-dependent manner ranging from 0.02 to 0.4 mg/mL, and it also relaxed the Ach-induced, 5-HT-induced andK+-induced contractions. SE shifted the Ca 2+concentration–response curves to right, similar to that caused by verapamil (0.025mM).
Conclusions: These findings indicated that SE played a spasmolytic role in gastrointestinal motility which was probably mediated through inhibition of muscarinic receptors, 5-HT receptors and calcium influx. All these results provide pharmacological basis for its clinical use in gastrointestinal tract.

Apocynin attenuates isoproterenol-induced myocardial injury and fibrogenesis

Li Liu, Jingang Cui, Qinbo Yang, Chenglin Jia, Minqi Xiong, et al.
Biochem Biophys Res Commun 449(2014)55–61
http://dx.doi.org/10.1016/j.bbrc.2014.04.157

Oxidative stress is mechanistically implicated in the pathogenesis of myocardial injury and the subsequent fibrogenic tissue remodeling. Therapies targeting oxidative stresss in the process of myocardial fibrogenesis are still lacking and thus remain as an active research area in myocardial injury management. The current study evaluated the effects of a NADPH oxidase inhibitor, apocynin, on the production of reactive oxygen species and  the development of myocardial fibrogenesis in isoproterenol (ISO)-induced myocardial injury mouse model. The results revealed a remarkable effect of apocynin on attenuating the development of myocardial necrotic lesions, inflammation and fibrogenesis. Additionally, the protective effects of apocynin against myocardial injuries were associated with suppressed expression of an array of genes implicated in inflammatory and fibrogenic responses. Our study thus provided for the first time the histopathological and molecular evidence supporting the therapeutic value of apocynin against the development of myocardial injuries, in particular, myocardial fibrogenesis, which will benefit the mechanism-based drug development targeting oxidative stress in preventing and/or treating related myocardial disorders.

Anti-tumor effect of Shu-gan-Liang-Xue decoction in breast cancer is related to the inhibition of aromatase and steroid sulfatase expression

Ning Zhou, Shu-Yan Han, Fei Zhou, Ping-ping Li
J Ethnopharmacol 154 (2014) 687–695
http://dx.doi.org/10.1016/j.jep.2014.04.045

Ethnopharmacological relevance: Shu-Gan-Liang-Xue Decoction (SGLXD), a traditional Chinese herbal formula used to ameliorate the hot flushes in breast cancer patients, was reported to have anti-tumor effect on breast cancer. Estrogen plays a critical role in the genesis and evolution of breast cancer. Aromatase and steroid sulfatase (STS) are key estrogen synthesis enzymes that predominantly contribute to the high local hormone concentrations. The present study was to evaluate the anti-tumor effect of SGLXD on estrogen receptor (ER) positive breast cancer celllineZR-75-1, and to investigate its underlying mechanisms both in vitro and in vivo.
Materials and methods: The anti-tumor activity of SGLXD in vitro was investigated using the MTT assay. The in vivo anti-tumor effect of SGLXD was evaluated in non-ovariectomized and ovariectomized athymic nude mice. The effect of SGLXD on enzymatic activity of aromatase and STS was examined using the dual-luciferase  reporter (DLR) based on bioluminescent measurements. Aromatase and STS protein level were assessed using Western blot assay. Results: SGLXD showed dose-dependent inhibitory effect on the proliferation of ZR-75-1 cells with IC50 value of 3.40 mg/mL. It also suppressed the stimulating effect on cell proliferation of testosterone and estrogen sulfates (E1S). Oral administration of 6 g/kg of SGLXD for 25 days resulted in a reduction in tumor volume in non-ovariectomized and ovariectomized nude mice. The bioluminescent measurements confirmed that SGLXD has a dual-inhibitory effect on the activity of aromatase and STS. Western blot assay demonstrated that the treatment of SGLXD resulted in a decrease in aromatase and STS protein levels both in vitro and in vivo. Conclusion: Our results suggested that SGLXD showed anti-tumor effect on breast cancer cells both in vitro and in vivo. The anti-tumor activity of SGLXD is related to inhibition of aromatase and STS via decreasing their expression. SGLXD may be considered as a novel treatment for ER positive breast cancer.

Cardioprotective effect of embelin on isoproterenol-induced myocardial injury in rats: Possible involvement of mitochondrial dysfunction and apoptosis

Bidya Dhar Sahu, Harika Anubolu, Meghana Koneru, et al.
Life Sciences 107 (2014) 59–67
http://dx.doi.org/10.1016/j.lfs.2014.04.035

Aims: Preventive and/or therapeutic interventions using natural products for ischemic heart disease have gained considerable attention world wide. This study investigated the cardioprotective effect and possible mechanism of embelin, a major constituent of EmbeliaribesBurm, using isoproterenol (ISO)-induced myocardial infarction model in rats.
Materials and methods: Rats were pretreated for three days with embelin (50mg/kg,p.o) before inducing myocardial injury by administration of ISO (85mg/kg) subcutaneously at aninterval of 24h for 2 consecutive days. Serum was analyzed for cardiac specific injury biomarkers, lipids and lipoprotein content. Heart tissues were isolated and were used for histopathology, antioxidant and mitochondrial respiratory enzyme activity assays and western blot analysis. Key findings: Results showed that pretreatment with embelin significantly decreased the elevated levels of serum specific cardiac injury biomarkers (CK-MB, LDH and AST), serum levels of lipids and lipoproteins and histopathological changes when compared to ISO-induced controls. Exploration of the underlying mechanisms of embelin action revealed that embelin pretreatment restored the myocardial mitochondrial respiratory enzyme activities (NADH dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and mitochondrial redox activity), strengthened antioxidant status and attenuated ISO-induced myocardial lipid peroxidation. Immunoblot analysis revealed that embelin interrupted mitochondria dependent apoptotic damage by increasing the myocardial expression of Bcl-2 and downregulating the expression of Bax, cytochrome c, cleaved-caspase-3&9 and PARP. Histopathology findings further strengthened the cardioprotective findings of embelin. Significance: Result suggested that embelin may have a potential benefit in preventing ischemic heart diseasel like myocardial infarction.

Effect of Matricaria chamomilla L. flower essential oil on the growth and ultrastructure of Aspergillus niger vanTieghem

Marziyeh Tolouee, Soheil Alinezhad, Reza Saberi, Ali Eslamifar, et al.
Intl J Food Microbiol 139 (2010) 127–133
http://dx.doi.org:/10.1016/j.ijfoodmicro.2010.03.032

The antifungal activity of Matricaria chamomilla L. flower essential oil was evaluated against Aspergillus niger with the emphasis on the plant’s mode of action at the electron microscopy level. A total of 21 compounds were identified in the plant oil using gaschromatography/massspectrometry(GC/MS) accounting for 92.86% of the oil composition. The main compounds identified were α-bisabolol (56.86%), trans-trans-farnesol (15.64%), cis-β-farnesene (7.12%), guaiazulene (4.24%), α-cubebene (2.69%), α-bisabololoxideA (2.19%) and chamazulene (2.18%). In the bioassay, A.niger was cultured on Potato Dextrose Broth medium in 6-well microplates in the presence of serial two fold concentrations of plant oil (15.62 to 1000 µg/mL) for 96h at 28°C. Based on the results obtained, A. niger growth was inhibited dose dependently with a maximum of ∼92.50% at the highest oil concentration. A marked retardation in conidial production by the fungus was noticed in relation to the inhibition of hyphal growth. The main changes of hyphae observed by transmission electron microscopy were disruption of cytoplasmic membranes and intracellular organelles, detachment of plasma membrane from the cell wall, cytoplasm depletion, and completed is organization of hyphal compartments. In scanning electron microscopy, swelling and deformation of hyphal tips, formation of short branches, and collapse of entire hyphae were the major changes observed. Morphological alterations might be due to the effect on cell permeability through direct interaction of M. chamomilla essential oil with the fungal plasma membrane. These findings indicate the potential of M. chamomilla L. essential oil in preventing fungal contamination and subsequent deterioration of stored food and other susceptible materials.

Related articles in Pharmaceutical Intelligence.

Plant-based Nutrition, Neutraceuticals and Alternative Medicine: Article Compilation the Journal PharmaceuticalIntelligence.com

https://pharmaceuticalintelligence.com/2014/09/01/compilation-of-lpi-articles-on-plant-based-nutrition-and-neutraceuticals-alternative-medicine/

What do you know about Plants and Neutraceuticals?

https://pharmaceuticalintelligence.com/2014/07/30/what-do-you-know-about-plants-and-neutraceuticals/

The Discovery and Properties of Avemar – Fermented Wheat Germ Extract: Carcinogenesis Suppressor

https://pharmaceuticalintelligence.com/2014/06/07/the-discovery-and-properties-of-avemar-fermented-wheat-germ-extract-carcinogenesis-suppressor/

Introduction to Metabolomics

https://pharmaceuticalintelligence.com/2014/10/21/introduction-to-metabolomics/

Summary of Genomics and Medicine: Role in Cardiovascular Diseases

https://pharmaceuticalintelligence.com/2014/01/06/summary-of-genomics-and-medicine-role-in-cardiovascular-diseases/

Natural Products Chemistry

https://pharmaceuticalintelligence.com/2015/01/27/natural-products-chemistry/

Diet and Diabetes

https://pharmaceuticalintelligence.com/2015/03/02/diet-and-diabetes/

An Overview of Clinical Microbiology, Classification, and Antimicrobial Resistance

https://pharmaceuticalintelligence.com/2015/01/17/an-overview-of-clinical-microbiology-classification-and-antimicrobial-resistance/

Diabetes Mellitus

https://pharmaceuticalintelligence.com/2014/10/24/diabetes-mellitus/

Mechanisms of Drug Resistance

https://pharmaceuticalintelligence.com/2014/10/09/mechanisms-of-drug-resistance/

Antioxidant Potential of “Maltese Mushroom” (Cynomorium coccineum)

https://pharmaceuticalintelligence.com/2013/01/27/antioxidant-potential-of-maltese-mushroom-cynomorium-coccineum/

Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets

https://pharmaceuticalintelligence.com/2012/10/22/advances-in-separations-technology-for-the-omics-and-clarification-of-therapeutic-targets/

Acute Lung Injury

https://pharmaceuticalintelligence.com/2015/02/26/acute-lung-injury/

The Vibrant Philly Biotech Scene: Focus on KannaLife Sciences and the Discipline and Potential of Pharmacognosy

Curator and Interviewer: Stephen J. Williams, Ph.D.

https://pharmaceuticalintelligence.com/2015/02/19/the-vibrant-philly-biotech-scene-focus-on-kannalife-sciences-and-the-discipline-and-potential-of-pharmacognosy/

Complex Models of Signaling: Therapeutic Implications

https://pharmaceuticalintelligence.com/2014/10/31/complex-models-of-signaling-therapeutic-implications/

Parasitic Plant Strangleweed Injects Host With Over 9,000 RNA Transcripts

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2014/09/02/parasitic-plant-strangleweed-injects-host-with-over-9000-rna-transcripts/

Health benefit of anthocyanins from apples and berries noted for men

https://pharmaceuticalintelligence.com/2014/07/06/health-benefit-of-anthocyanins-from-apples-and-berries-noted-for-men/

Requiem for Palliative Cardiology: The Voice of Dr. Esselstyn on Plant-Based Nutrition

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2014/02/02/requiem-for-palliative-cardiology-the-voice-of-dr-esselstyn-on-plant-based-nutrition/

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Depth Underwater and Underground

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

Introduction

Deep diving for mammals is dangerous for humans and land based animals for too long, and it has dangerous consequences, most notable in nitrogen emboli  with very deep underwater diving. Other mammals live in water and have adapted to a water habitat.  This is another topic that needs further exploration.

Deep diving has different meanings depending on the context. Even in recreational diving the meaning may vary:

In recreational diving, a depth below about 30 metres (98 ft), where nitrogen narcosis becomes a significant hazard for most divers, may be considered a “deep dive”

In technical diving, a depth below about 60 metres (200 ft) where hypoxic breathing gas becomes necessary to avoid oxygen toxicity may be considered a “deep dive”.

Early experiments carried out by Comex S.A. (Compagnie maritime d’expertises) using hydrox and trimix attained far greater depths than any recreational technical diving. One example being the Comex Janus IV open-sea dive to 501 metres (1,644 ft) in 1977. The open-sea diving depth record was achieved in 1988 by a team of Comex divers who performed pipe line connection exercises at a depth of 534 metres (1,752 ft) in the Mediterranean Sea as part of the Hydra 8 program. These divers needed to breathe special gas mixtures because they were exposed to very high ambient pressure (more than 50 times atmospheric pressure).

Then there is the adaptation to the water habitat as a living environment. The two main types of aquatic ecosystems are marine ecosystems and freshwater ecosystems.

http://en.wikipedia.org/wiki/Deep_diving

Marine ecosystems are part of the earth’s aquatic ecosystem. The habitats that make up this vast system range from the productive nearshore regions to the barren ocean floor. The marine waters may be fully saline, brackish or nearly fresh. The saline waters have a salinity of 35-50 ppt (= parts per thousand). The freshwater has a salinity of less than 0.5 ppt. The brackish water lies in between these 2. Marine habitats are situated from the coasts, over the continental shelf to the open ocean and deep sea. The ecosystems are sometimes linked with each other and are sometimes replacing each other in other geographical regions. The reason why habitats differ from another is because of the physical factors that influence the functioning and diversity of the habitats. These factors are temperature, salinity, tides, currents, wind, wave action, light and substrate.

Marine ecosystems are home to a host of different species ranging from planktonic organisms that form the base of the marine food web to large marine mammals. Many species rely on marine ecosystems for both food and shelter from predators. They are very important to the overall health of both marine and terrestrial environments. Coastal habitats are those above the spring high tide limit or above the mean water level in non-tidal waters.  They are close to the sea and include habitats such as coastal dunes and sandy shores, beaches , cliffs and supralittoral habitats. Coastal habitats alone account for approximately 30% of all marine biological productivity.

http://www.marbef.org/wiki/marine_habitats_and_ecosystems

All plant and animal life forms are included from the microscopic picoplankton all the way to the majestic blue whale, the largest creature in the sea—and for that matter in the world. It wasn’t until the writings of Aristotle from 384-322 BC that specific references to marine life were recorded. Aristotle identified a variety of species including crustaceans, echinoderms, mollusks, and fish.
Today’s classification system was developed by Carl Linnaeus external link as an important tool for use in the study of biology and for use in the protection of biodiversity. Without very specific classification information and a naming system to identify species’ relationships, scientists would be limited in attempts to accurately describe the relationships among species. Understanding these relationships helps predict how ecosystems can be altered by human or natural factors.

Preserving biodiversity is facilitated by taxonomy. Species data can be better analyzed to determine the number of different species in a community and to determine how they might be affected by environmental stresses. Family, or phylogenetic, trees for species help predict environmental impacts on individual species and their relatives.

http://marinebio.org/oceans/marine-taxonomy/

For generations, whales and other marine mammals have intrigued humans. 2,400 years ago, Aristotle, a Greek scientist and philosopher, recognized that whales are mammals, not fish, because they nurse their young and breathe air like other mammals. There are numerous myths and legends surrounding marine mammals. The Greeks believed that killing a dolphin was as bad as murdering a human. An Amazon legend said that river dolphins came to shore dressed as men to woo pretty girls during fiestas. During the Middle Ages, there were numerous legends surrounding the narwhals’ amazing tusk, which was thought to have come from the unicorn.

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Marine mammals evolved from their land dwelling ancestors over time by developing adaptations to life in the water. To aid swimming, the body has become streamlined and the number of body projections has been reduced. The ears have shrunk to small holes in size and shape. Mammary glands and sex organs are not part of the external physiology, and posterior (hind) limbs are no longer present.

Mechanisms to prevent heat loss have also been developed. The cylindrical body shape with small appendages reduces the surface area to volume ratio of the body, which reduces heat loss. Marine mammals also have a counter current heat exchange mechanism created by convergent evolution external link where the heat from the arteries is transferred to the veins as they pass each other before getting to extremities, thus reducing heat loss. Some marine mammals also have a thick layer of fur with a water repellent undercoat and/or a thick layer of blubber that can’t be compressed. The blubber provides insulation, a food reserve, and aids with buoyancy. These heat loss adaptations can also lead to overheating for animals that spend time out of the water. To prevent overheating, seals or sea lions will swim close to the surface with their front flippers waving in the air. They also flick sand onto themselves to keep the sun from directly hitting their skin. Blood vessels can also be expanded to act as a sort of radiator.

One of the major behavioral adaptations of marine mammals is their ability to swim and dive. Pinnipeds swim by paddling their flippers while sirenians and cetaceans move their tails or flukes up and down.

Some marine mammals can swim at relatively high speeds. Sea lions swim up to 35 kph and orcas can reach 50 kph. The fastest marine mammal, however, is the common dolphin, which reaches speeds up to 64 kph. While swimming, these animals take very quick breaths. For example, fin whales can empty and refill their huge lungs in less than 2 seconds. Marine mammals’ larynx and esophagus close automatically when they open their mouths to catch prey during dives. Oxygen is stored in hemoglobin in the blood and in myoglobin in the muscles. The lungs are also collapsible so that air is pushed into the windpipe preventing excess nitrogen from being absorbed into the tissues. Decreasing pressure can cause excess nitrogen to expand in the tissues as animals ascend to shallower depths, which can lead to decompression sickness,  aka “the bends.” Bradycardia, the reduction of heart rate by 10 to 20%, also takes place to aid with slowing respiration during dives and the blood flow to non-essential body parts. These adaptations allow sea otters to stay submerged for 4 to 5 minutes and dive to depths up to 55 m. Pinnipeds can often stay down for 30 minutes and reach average depths of 150-250 m. One marine mammal with exceptional diving skills is the Weddell seal, which can stay submerged for at least 73 minutes at a time at depths up to 600 m. The length and depth of whale dives depends on the species. Baleen whales feed on plankton near the surface of the water and have no need to dive deeply so they are rarely seen diving deeper than 100 m external link. Toothed whales seek larger prey at deeper depths and some can stay down for hours at depths of up to 2,250 m external link.

http://marinebio.org/oceans/marine-mammals/

Human Experience

Albert Behnke: Nitrogen Narcosis

Casey A. Grover and David H. Grover
The Journal of Emergency Medicine, 2014; 46(2):225–227
http://dx.doi.org/10.1016/j.jemermed.2013.08.080

As early as 1826, divers diving to great depths noted that descent often resulted in a phenomenon of intoxication and euphoria. In 1935, Albert Behnke discovered nitrogen as the cause of this clinical syndrome, a condition now known as nitrogen narcosis. Nitrogen narcosis consists of the development of euphoria, a false sense of security, and impaired judgment upon underwater descent using compressed air below 34 atmospheres (99 to 132 feet). At greater depths, symptoms can progress to loss of consciousness. The syndrome remains relatively unchanged in modern diving when compressed air is used. Behnke’s use of non-nitrogencontaining gas mixtures subsequent to his discovery during the 1939 rescue of the wrecked submarine USS Squalus pioneered the use of non-nitrogencontaining gas mixtures, which are used by modern divers when working at great depth to avoid the effects of nitrogen narcosis.

Behnke’s first duty station as a licensed physician was as assistant medical officer for Submarine Division 20 in San Diego, which was then commanded by one of the Navy’s rising stars, Captain Chester W. Nimitz of World War II fame.
In this setting, Dr. Behnke spent his free time constructively by learning to dive, using the traditional ‘‘hard-hat’’ gear aboard the USS Ortalon, a submarine rescue vessel to which he also rotated. Diving was not a notable specialty of the Navy at the time, and the service was slow in developing the infrastructure for it. Dr. Behnke devoted his efforts to research on the topic of diving medicine, as well as developing a more sound understanding of the biophysics of diving. In 1932, he wrote a letter to the Surgeon General describing some of his observations on arterial gas embolism, which earned him some accolades from the Navy and resulted in his transfer to Harvard’s School of Public Health as a graduate fellow. After 2 years at Harvard, the Navy assigned duty to Dr. Behnke at the Navy’s submarine escape training tower at Pearl Harbor. He worked extensively here on developing techniques for rescuing personnel from disabled submarines on the sea floor. In 1937, he was one of three Navy physicians assigned to the Navy’s Experimental Diving Unit. This team worked on improving the rescue system, plus updating the diving recompression tables originally developed by the British in 1908.

The intoxicating effects of diving were first described by a French physician named Colladon in 1826, who reported that descent in a diving bell resulted in his feeling a ‘‘state of excitement as though I had drunk some alcoholic liquor’’.
The etiology of this phenomenon remained largely unknown until the 1930s, when the British military researcher Damant again highlighted the issue, and reported very unpredictable behavior in his divers during descents as deep as 320 feet during the British Admiralty Deep Sea diving trials. Two initial theories arose as to the etiology for this effect, the first being from psychological causes by Hill and Phillip in 1932, and the second being from oxygen toxicity by Haldane in 1935.

Dr. Behnke and his colleagues at the Harvard School of Public Health had another idea as to the etiology of this phenomenon. In 1935, based on observation of individuals in experiments with a pressure chamber, Dr. Behnke published an article in the American Journal of Physiology in which he posited that nitrogen was the etiology of the intoxicating effects of diving.

Nitrogen narcosis, described as ‘‘rapture of the deep’’ by Jacques Cousteau, still remains a relatively common occurrence in modern diving, despite major advances in diving technology since Behnke’s initial description of the pathophysiologic cause of the condition in 1935. The development of symptoms of this condition varies from diver to diver, but usually begins when a depth of 4 atmospheres (132 feet) is reached in divers using compressed air. More sensitive divers can develop symptoms at only 3 atmospheres (99 feet), and other divers may not be affected up to depths as high as 6 atmospheres (198 feet). Interestingly, tolerance to nitrogen narcosis can be developed by frequent diving and exposure to the effects of compressed air at depth.

  1. Acott C. A brief history of diving and decompression illness. SPUMS J 1999;29:98–109.
    2. Bornmann R. Dr. Behnke, founder of UHMS, dies. Pressure 1992; 21:14.
    3. Behnke AR, Thomson RM, Motley P. The psychologic effects from breathing air at 4 atmospheric pressures. Am J Physiol 1935; 112:554–8.
    4. Behnke AR, Johnson FS, Poppen JR, Motley P. The effect of oxygen on man at pressures from 1 to 4 atmospheres. AmJ Physiol 1934; 110:565–72.

Exhaled nitric oxide concentration and decompression-induced bubble formation: An index of decompression severity in humans?

J.-M. Pontier, Buzzacott, J. Nastorg, A.T. Dinh-Xuan, K. Lambrechts
Nitric Oxide 39 (2014) 29–34
http://dx.doi.org/10.1016/j.niox.2014.04.005

Introduction: Previous studies have highlighted a decreased exhaled nitric oxide concentration (FE NO) in divers after hyperbaric exposure in a dry chamber or following a wet dive. The underlying mechanisms of this decrease remain however unknown. The aim of this study was to quantify the separate effects of submersion, hyperbaric hyperoxia exposure and decompression-induced bubble formation on FE NO after a wet dive.
Methods: Healthy experienced divers (n = 31) were assigned to either

  • a group making a scuba-air dive (Air dive),
  • a group with a shallow oxygen dive protocol (Oxygen dive) or

a group making a deep dive breathing a trimix gas mixture (deep-dive).
Bubble signals were graded with the KISS score. Before and after each dive FE NO values were measured using a hand-held electrochemical analyzer.
Results: There was no change in post-dive values of FE NO values (expressed in ppb = parts per billion) in the Air dive group (15.1 ± 3.6 ppb vs. 14.3 ± 4.7 ppb, n = 9, p = 0.32). There was a significant decrease in post-dive values of FE NO in the Oxygen dive group (15.6 ± 6 ppb vs. 11.7 ± 4.7 ppb, n = 9, p = 0.009). There was an even more pronounced decrease in the deep dive group (16.4 ± 6.6 ppb vs. 9.4 ± 3.5 ppb, n = 13, p < 0.001) and a significant correlation between KISS bubble score >0 (n = 13) and percentage decrease in post-dive FE NO values (r = -0.53, p = 0.03). Discussion: Submersion and hyperbaric hyperoxia exposure cannot account entirely for these results suggesting the possibility that, in combination, one effect magnifies the other. A main finding of the present study is a significant relationship between reduction in exhaled NO concentration and dive-induced bubble formation. We postulate that exhaled NO concentration could be a useful index of decompression severity in healthy human divers.

Brain Damage in Commercial Breath-Hold Divers

Kiyotaka Kohshi, H Tamaki, F Lemaıtre, T Okudera, T Ishitake, PJ Denoble
PLoS ONE 9(8): e105006 http://dx.doi.org:/10.1371/journal.pone.0105006

Background: Acute decompression illness (DCI) involving the brain (Cerebral DCI) is one of the most serious forms of diving related injuries which may leave residual brain damage. Cerebral DCI occurs in compressed air and in breath-hold divers, likewise. We conducted this study to investigate whether long-term breath-hold divers who may be exposed to repeated symptomatic and asymptomatic brain injuries, show brain damage on magnetic resonance imaging (MRI).
Subjects and Methods: Our study subjects were 12 commercial breath-hold divers (Ama) with long histories of diving work in a district of Japan. We obtained information on their diving practices and the presence or absence of medical problems, especially DCI events. All participants were examined with MRI to determine the prevalence of brain lesions.
Results: Out of 12 Ama divers (mean age: 54.965.1 years), four had histories of cerebral DCI events, and 11 divers demonstrated ischemic lesions of the brain on MRI studies. The lesions were situated in the cortical and/or subcortical area (9 cases), white matters (4 cases), the basal ganglia (4 cases), and the thalamus (1 case). Subdural fluid collections were seen in 2 cases. Conclusion: These results suggest that commercial breath-hold divers are at a risk of clinical or subclinical brain injury which may affect the long-term neuropsychological health of divers.

Decompression illness

Richard D Vann, Frank K Butler, Simon J Mitchell, Richard E Moon
Lancet 2010; 377: 153–64

Decompression illness is caused by intravascular or extravascular bubbles that are formed as a result of reduction in environmental pressure (decompression). The term covers both arterial gas embolism, in which alveolar gas or venous gas emboli (via cardiac shunts or via pulmonary vessels) are introduced into the arterial circulation, and decompression sickness, which is caused by in-situ bubble formation from dissolved inert gas. Both syndromes can occur in divers, compressed air workers, aviators, and astronauts, but arterial gas embolism also arises from iatrogenic causes unrelated to decompression. Risk of decompression illness is
affected by immersion, exercise, and heat or cold. Manifestations range from itching and minor pain to neurological symptoms, cardiac collapse, and death. First aid treatment is 100% oxygen and definitive treatment is recompression to increased pressure, breathing 100% oxygen. Adjunctive treatment, including fluid administration and prophylaxis against venous thromboembolism in paralyzed patients, is also recommended. Treatment is, in most cases, effective although residual deficits can remain in serious cases, even after several recompressions.

Bubbles can have mechanical, embolic, and biochemical effects with manifestations ranging from trivial to fatal. Clinical manifestations can be caused by direct effects from extravascular (autochthonous) bubbles such as mechanical distortion of tissues causing pain, or vascular obstruction causing stroke-like signs and symptoms. Secondary effects can cause delayed symptom onset up to 24 h after surfacing. Endothelial damage by intravascular bubbles can cause capillary leak, extravasation of plasma, and haemoconcentration. Impaired endothelial function, as measured by decreased effects of vasoactive compounds, has been reported in animals and might occur in man. Hypotension can occur in severe cases. Other effects include platelet activation and deposition, leucocyte-endothelial adhesion, and possibly consequences of vascular occlusion believed to occur in thromboembolic stroke such as ischaemia-reperfusion injury, and apoptosis.

Classification of initial and of all eventual manifestations of decompression illness in 2346 recreational diving accidents reported to the Divers Alert Network from 1998 to 2004 For all instances of pain, 58% consisted of joint pain, 35% muscle pain, and 7% girdle pain. Girdle pain often portends spinal cord involvement. Constitutional symptoms included headache, lightheadedness, inappropriate fatigue, malaise, nausea or vomiting, and anorexia. Muscular discomfort included stiffness, pressure, cramps, and spasm but excluded pain. Pulmonary manifestations included dyspnoea and cough.

Other than depth and time, risk of decompression sickness is affected by other factors that affect inert gas exchange and bubble formation, such as immersion (vs dry hyperbaric chamber exposure), exercise, and temperature. Immersion decreases venous pooling and increases venous return and cardiac output. Warm environments improve peripheral perfusion by promoting vasodilation, whereas cool temperatures decrease perfusion through vasoconstriction. Exercise increases both peripheral perfusion and temperature. The effect of environmental conditions on risk of decompression sickness is dependent on the phase of the pressure exposure. Pressure, exercise, immersion, or a hot environment increase inert gas uptake and risk of decompression sickness. During decom-pression these factors increase inert gas elimination and therefore decrease the risk of decompression sickness. Conversely, uptake is reduced during rest or in a cold environment, hence a diver resting in a cold environment on the bottom has decreased risk of decompression sickness. Rest or low temperatures during decompression increase the risk. If exercise occurs after decompression when super-saturation is present, bubble formation increases and risk of decompression sickness rises.

Exercise at specific times before a dive can decrease the risk of serious decompression sickness in animals and incidence of venous gas emboli in both animals and man. The mechanisms of these effects are unknown but might involve modulation of nitric oxide production and effects on endothelium. Venous gas emboli and risk of decompression sickness increase slightly with age and body-mass index.

Arterial gas embolism should be suspected if a diver has a new onset of altered consciousness, confusion, focal cortical signs, or seizure during ascent or within a few minutes after surfacing from a compressed gas dive.

If the diver spends much time at depth and might have absorbed substantial inert gas before surfacing, arterial gas embolism and serious decompression sickness can coexist, and in such cases, spinal cord manifestations can predominate. Other organ systems, such as the heart, can also be affected, but the clinical diagnosis of gas embolism is not reliable without CNS manifestations. Arterial gas embolism is rare in altitude exposure; if cerebral symptoms occur after altitude exposure, the cause is usually decompression sickness.

Nondermatomal hypoaesthesia and truncal ataxia are common in neurological decompression sickness and can be missed by cursory examination. Pertinent information includes level of consciousness and mental status, cranial nerve function, and motor strength. Coordination can be affected disproportionately, and abnormalities can be detected by assessment of finger-nose movement, and, with eyes open and closed, ability to stand and walk and do heel-toe walking backwards and forwards. Many of these simple tests can be done on the scene by untrained companions.

Panel: Differential diagnosis of decompression illness
Inner-ear barotrauma
Middle-ear or maxillary sinus overinfl ation
Contaminated diving gas and oxygen toxic effects
Musculoskeletal strains or trauma sustained before, during, or after diving
Seafood toxin ingestion (ciguatera, pufferfish, paralytic shellfish poisoning)
Immersion pulmonary edema
Water aspiration
Decompression chamber

Decompression chamber

Decompression chamber. fluidic or pneumatic ventilator is shown at the left. The infusion pump is contained within a plastic cover, in which 100% nitrogen is used to decrease the fi re risk in the event of an electrical problem. The monitor screen is outside the chamber and can be seen through the viewing port. Photo from Duke University Medical Center, with permission.

Long-term outcomes of 69 divers with spinal cord decompressionsickness, by manifestation
n %
No residual symptoms 34 49·3
Any residual symptom 35 50·7
Mild paraesthesias, weakness, or pain 14 20·3
Some impairment of daily activities 21 30·4
Difficulty walking 11 15·9
Impaired micturition 13 18·8
Impaired defecation 15 21·7
Impaired sexual function 15 21·7

Decompression illness occurs in a small population but is an international problem that few physicians are trained to recognise or manage. Although its manifestations are often mild, the potential for permanent injury exists in severe cases, especially if unrecognised or inadequately treated. Emergency medical personnel should be aware of manifestations of decompression illness in the setting of a patient with a history of recent diving or other exposure to substantial pressure change, and should contact an appropriate consultation service for advice.

Diving Medicine: Contemporary Topics and Their Controversies

Michael B. Strauss and Robert C. Borer, Jr
Am J Emerg Med 2001; 19:232-238
http://dx.doi.org:/10.1053/ajem.2001.22654

SCUBA diving is a popular recreational sport. Although serious injuries occur infrequently, when they do knowledge of diving medicine and/or where to obtain appropriate consultation is essential. The emergency physician is likely to be the first physician contact the injured diver has. We discuss 8 subjects
in diving medicine which are contemporary, yet may have controversies associated with them. From this information the physician dealing primarily with the injured diver will have a basis for understanding and managing, as
well as where to find additional help, for his/her patients’ diving injuries.

Over the past 10 years, new knowledge and equipment improvements have made diving safer and more enjoyable. Estimates of actively participating sports divers show a striking increase over this time interval while the number of SCUBA diving deaths annually has remained nearly level at approximately 100. A further indicator of recreational diving safety is that reflected in the nearly constant number of diving injuries (1000 per annum) over the most recent 5 reported years, or approximately 0.53 to 3.4 incidents/10,000 dives.

Divers Alert Network.
The Divers Alert Network (DAN) is a nonprofit organization directed and staffed by experts in the specialty of diving medicine.6 DAN provides immediate consultation for both divers and physicians in the diagnosis and initial management of diving injuries. This 24-hour service is available free world-wide through a dedicated emergency telephone line: 1-919-684-4326. The DAN staff will also identify the nearest appropriate recompression treatment facility and knowledgeable physicians for an expedient referral. General diving medical inquiries can be answered during normal weekday hours either through an information telephone line: 1-919-684-2948 or through an interactive web site http://www.diversalertnetwork.org.

Use of 100% Oxygen for Initial, on the Scene, Management of Diving Accidents
The breathing of pure oxygen is crucial for the initial management of the diving related problems of arterial gas embolism (AGE), decompression sickness (DCS), pulmonary barotrauma (thoracic squeeze), aspiration pneumonitis, and hypoxic encephalopathy associated with near drowning. In 1985, Dick reported that in many cases the neurologic symptoms of AGE and DCS were resolved with the immediate breathing of pure oxygen on the surface. The breathing of pure oxygen reduces bubble size by increasing the differential pressure for the inert gas to diffuse out of the bubble and it also speeds the washout of inert gas from body tissues. The early elimination of the bubble prevents hypoxia and the interaction of the bubble with the blood vessel lining. This interaction leads to secondary problems of capillary leak, bleeding, inflammation, ischemia, and cell death. These secondary problems are the reasons not all DCS symptoms resolve with recompression chamber treatment. The immediate use of pure oxygen for the medical management of these diving problems is analogous to the use of cardiopulmonary resuscitation for the witnessed cardiac arrest; the sooner initiated the better the results.

Diving Education

Medical Fitness for Diving

Asthma has the potential risk for AGE. Neuman reviewed the subject of asthma and diving. He and his coauthors recommend that asthmatics who are asymptomatic, not on medications and have no exercised induced abnormality on pulmonary function studies be allowed to dive.

Conditions leading to loss of consciousness, such as insulin dependent diabetes and epilepsy, can result in drowning. Carefully controlled diving studies in diabetics, who are free from complications, are now defining the safe requirements for diving. Epilepsy remains as a disqualification except in individuals with a history of febrile seizures ending prior to 5 years of age.

Availability of Hyperbaric Oxygen Treatment Facilities

The availability of these chambers makes it possible for divers who become symptomatic after SCUBA diving to readily receive recompression treatment. This is important because the closer the initiation of recompression treatment to the onset of DCS (and AGE) signs and symptoms, the greater the likelihood of full recovery.

Improved Diving Equipment

Mixed and Rebreather Gas Diving
Mixed gas diving involves changing the breathing gas from air which has 20% oxygen to higher oxygen percentages (nitrox). As the amount of oxygen is increased in the gas mixture, the amount of the inert gas (nitrogen) is reduced. With oxygen enriched air there is less tissue deposition of inert gas per unit of time under water for any given depth. However, because of increased oxygen partial pressures, the seizure threshold for oxygen toxicity is lowered. For normal sports diving activities, oxygen toxicity with mixed gas diving is only a theoretical concern.

Decompression Illness is More Than Bubbles

When AGE occurs, DCS symptoms may be concurrent or appear during or after recompression treatment even though the decompression tables were not violated on the dive. When DCS occurs in this situation it appears resistant to recompression treatment (Neuman) perhaps because of the inflammatory reaction generated by the bubble-blood vessel interaction from the AGE. In cases of DCI where components of both DCS and AGE are suspected, the diver should be observed for a minimum of 24 hours after the recompression treatment is completed for the delayed onset of DCS.

No theory of DCS discounts the primary role of bubbles in this condition. However, new information suggests that there are precursors to bubble formation and post-bubbling events that occur as a consequence of the bubbles. As mentioned earlier, venous gas emboli are a common occurrence diving ascent and ordinarily are filtered out harmlessly by the lungs. Precursors to DCS include stasis, dehydration and too rapid of ascents. These conditions allow the ubiquitous VGE to enlarge, coalesce and occlude the venous side of the circulation. Massive venous bubbling to the lungs can cause pulmonary vessel obstruction described as the chokes. If right to left shunts occur in the heart, VGE can become AGE to the brain. If the arterial flow is slow enough and/or the gradients large enough, autochthonus (ie, spontaneous) bubbles can form in the arterial circulation and lead to any of the consequences of AGE. In such situations it could be difficult to determine whether the DCI event was from AGE or DCS even after careful analysis of the dive profile. Hollenbeck’s model for diving paraplegia includes the setting of venous stasis (Batson’s plexus of veins) in the spinal canal, bubble formation, bubble enlargement possibly from off gassing of the spinal cord, blood vessel occlusion, and venous side infarctions of the spinal cord.
Contemporary Management of DCS

Problem Intervention Effect
Bubble Recompression
with HBO
Reduce bubble size
1. Washout inert gas.
2. Change bubble composition by diffusion.
Stasis and dehydration Hydration: oral fluids if alert, IV fluids otherwise. Improve blood flow.
InflammationCell Ischemia ? Anti-inflammatory medicationsHBO Reduce interaction between bubble and blood vessel endothelium.
Improve oxygen availability to hypoxic tissues, reduce edema and also reduces the interaction between bubble and blood vessel endothelium.

.

Conclusions

We anticipate that in the future there will be further improvements for the safety and enjoyment of the recreational SCUBA diver. For example, the dive computer of the future will be able to individualize dive profiles for different personal medical parameters such as age, body composition and fitness level. Diver locators could quickly target a missing diver and save time and gas consumption as well as prevent serious diving mishaps. Drugs may be developed that would minimize the effect of bubbles interacting with body tissues and prevent DCS and AGE.

Extracorporeal membrane oxygenation therapy for pulmonary decompression illness

Yutaka Kondo, Masataka Fukami and Ichiro Kukita
Kondo et al. Critical Care 2014; 18:438 http://ccforum.com/content/18/3/438/10.1186/cc13935

Pulmonary decompression illness is rarely observed in clinical settings, and most patients die prior to hospitalization. We administered ECMO therapy to rescue a patient, even though this therapy has rarely been reported with good outcome in patients with decompression illness. In addition, we had to select venovenous ECMO even with the patient showing right ventricular failure. A lot of physicians may select venoarterial ECMO if the patient shows right ventricular failure, but the important physiological mechanism of pulmonary decompression illness is massive air embolism in the pulmonary arteries, and the bubbles diminish within the first 24 hours. The management of decompression illness therefore differs substantially from the usual right-sided heart failure.

Extremes of barometric pressure

Jane E Risdall, David P Gradwell
Anaesthesia and Intensive Care Medicine 16:2
Ascent to elevated altitude, commonly achieved through flight, by climbing or by residence in highland regions, exposes the individual to reduced ambient pressure. Although there are physical manifestations of this exposure as a consequence of Boyle’s law, the primary physiological challenge is of hypobaric hypoxia. The acute physiological and longer-term adaptive responses of the cardiovascular, respiratory, hematological and neurological systems to altitude are described, together with an outline of the presentation and management of acute mountain sickness, high-altitude pulmonary edema and high-altitude cerebral edema. While many millions experience modest exposure to altitude as a result of flight in pressurized aircraft, fewer individuals are exposed to increased ambient pressure. The pressure changes during diving and hyperbaric exposures result in greater changes in gas load and gas toxicity. Physiological effects include the consequences of increased work of breathing and redistribution of circulating volume. Neurological manifestations may be the direct result of pressure or a consequence of gas toxicity at depth. Increased tissue gas loads may result in decompression illness on return to surface or subsequent ascent in flight.

  • understand the physical effects of changes in ambient pressure and the physiological consequences on the cardiovascular respiratory and neurological systems
  • gain an awareness that exposure to reduced ambient pressure produces both acute and more chronic effects, with differing signs, symptoms and time to onset at various altitudes
  • develop an awareness of the toxic effects of ‘inert’ gases at increased ambient pressures and the pathogenesis and management of decompression illness

Decompression illness According to Henry’s law, at a constant temperature the amount of gas which dissolves in a liquid is proportional to the pressure of that gas or its partial pressure, if it is part of a mixture of gases. Breathing gases at increased ambient pressure will increase the amount of each gas dissolved in the fluid phases of body tissues. On ascent this excess gas has to be given up. If the ascent is controlled at a sufficiently slow rate, elimination will be via the respiratory system. If the ascent is too fast, excess gas may come out of solution and form free bubbles in the tissues or circulation. Bubbles may contain any of the gases in the breathing mixture, but it is the presence of inert gas bubbles (nitrogen or helium) that are thought most likely to give rise to problems, since the elimination of excess oxygen is achieved by metabolism as well as ventilation. These bubbles may act as venous emboli or may trigger inflammatory tissue responses giving rise to symptoms of decompression illness (DCI). Signs and symptoms of DCI may appear up to 48 hours after exposure to increased ambient pressure and include joint pains, motor and sensory deficits, dyspnoea, cough and skin rashes.

Neurological effects of deep diving

Marit Grønning, Johan A. Aarli
Journal of the Neurological Sciences 304 (2011) 17–21
http://dx.doi.org:/10.1016/j.jns.2011.01.021

Deep diving is defined as diving to depths more than 50 m of seawater (msw), and is mainly used for occupational and military purposes. A deep dive is characterized by the compression phase, the bottom time and the decompression phase. Neurological and neurophysiologic effects are demonstrated in divers during the compression phase and the bottom time. Immediate and transient neurological effects after deep dives have been shown in some divers. However, the results from the epidemiological studies regarding long term neurological effects from deep diving are conflicting and still not conclusive.

Possible immediate neurological effects of deep diving
Syndrome Pressure
Hyperoxia/oxygen seizures >152 kPa (5 msw)
HypoxiaHypercapnia
Nitrogen narcosis >354 kPa (25 msw)
High pressure nervous syndrome >1.6 MPa (150 msw)
Neurological decompression sickness

Neurological effects have been demonstrated, both clinically and neurophysiologically in divers during the compression phase and the bottom time. Studies of divers before and after deep dives have shown immediate and transient neurological effects in some divers. However, the results from the epidemiological and clinical studies regarding long term neurological effects from deep diving are conflicting and still not conclusive. Prospective clinical studies with sufficient power and sensitivity are needed to solve this important issue.

Today deep diving to more than 100 msw is routinely performed globally in the oil- and gas industry. In the North Sea remote underwater intervention and maintenance is performed by the use of remotely operated vehicles (ROV), both in conjunction to and as an alternative to manned underwater operations. There will, however, always be a need for human divers in the technically more advanced underwater operations and for contingency repair operations.

P300 latency indexes nitrogen narcosis

Barry Fowler, Janice Pogue and Gerry Porlier
Electroencephalography, and clinical Neurophysiology, 1990, 75:221-229

This experiment investigated the effects of nitrogen narcosis on reaction time (RT) and P300 latency and amplitude, Ten subjects breathed either air or a non-narcotic 20% oxygen-80% helium (heliox) mixture in a hyperbaric chamber at 6.5, 8.3 and 10 atmospheres absolute (ATA), The subjects responded under controlled accuracy conditions to visually presented male or female names in an oddball paradigm. Single-trial analysis revealed a strong relationship between RT and P300 latency, both of which were slowed in a dose-related manner by hyperbaric air but not by heliox. A clear-cut dose-response relationship could not be established for P300 amplitude. These results indicate that P300 latency indexes nitrogen narcosis and are interpreted as support for the slowed processing model of inert gas narcosis.

Adaptation to Deep Water Habitat

Effects of hypoxia on ionic regulation, glycogen utilization and antioxidative ability in the gills and liver of the aquatic air-breathing fish Trichogaster microlepis

Chun-Yen Huang, Hui-Chen Lina, Cheng-Huang Lin
Comparative Biochemistry and Physiology, Part A 179 (2015) 25–34
http://dx.doi.org/10.1016/j.cbpa.2014.09.001

We examined the hypothesis that Trichogaster microlepis, a fish with an accessory air-breathing organ, uses a compensatory strategy involving changes in both behavior and protein levels to enhance its gas exchange ability. This compensatory strategy enables the gill ion-regulatory metabolism to maintain homeostasis during exposure to hypoxia. The present study aimed to determinewhether ionic regulation, glycogen utilization and antioxidant activity differ in terms of expression under hypoxic stresses; fish were sampled after being subjected to 3 or 12 h of hypoxia and 12 h of recovery under normoxia. The air-breathing behavior of the fish increased under hypoxia. No morphological modification of the gills was observed. The expression of carbonic anhydrase II did not vary among the treatments. The Na+/K+-ATPase enzyme activity did not decrease, but increases in Na+/K+-ATPase protein expression and ionocyte levels were observed. The glycogen utilization increased under hypoxia as measured by glycogen phosphorylase protein expression and blood glucose level, whereas the glycogen content decreased. The enzyme activity of several components of the antioxidant system in the gills, including catalase, glutathione peroxidase, and superoxidase dismutase, increased in enzyme activity. Based on the above data, we concluded that T. microlepis is a hypoxia-tolerant species that does not exhibit ion-regulatory suppression but uses glycogen to maintain energy utilization in the gills under hypoxic stress. Components of the antioxidant system showed increased expression under the applied experimental treatments.

Divergence date estimation and a comprehensive molecular tree of extant cetaceans

Michael R. McGowen , Michelle Spaulding, John Gatesy
Molecular Phylogenetics and Evolution 53 (2009) 891–906
http://dx.doi.org/10.1016/j.ympev.2009.08.018

Cetaceans are remarkable among mammals for their numerous adaptations to an entirely aquatic existence, yet many aspects of their phylogeny remain unresolved. Here we merged 37 new sequences from the nuclear genes RAG1 and PRM1 with most published molecular data for the group (45 nuclear loci, transposons, mitochondrial genomes), and generated a supermatrix consisting of 42,335 characters. The great majority of these data have never been combined. Model-based analyses of the supermatrix produced a solid, consistent phylogenetic hypothesis for 87 cetacean species. Bayesian analyses corroborated odontocete (toothed whale) monophyly, stabilized basal odontocete relationships, and completely resolved branching events within Mysticeti (baleen whales) as well as the problematic speciose clade Delphinidae (oceanic dolphins). Only limited conflicts relative to maximum likelihood results were recorded, and discrepancies found in parsimony trees were very weakly supported. We utilized the Bayesian supermatrix tree to estimate divergence dates among lineages using relaxed-clock methods. Divergence estimates revealed rapid branching of basal odontocete lineages near the Eocene–Oligocene boundary, the antiquity of river dolphin lineages, a Late Miocene radiation of balaenopteroid mysticetes, and a recent rapid radiation of Delphinidae beginning [1]10 million years ago. Our comprehensive,  time calibrated tree provides a powerful evolutionary tool for broad-scale comparative studies of Cetacea.

Mitogenomic analyses provide new insights into cetacean origin and evolution

Ulfur Arnason, Anette Gullberg, Axel Janke
Gene 333 (2004) 27–34
http://dx.doi.org:/10.1016/j.gene.2004.02.010

The evolution of the order Cetacea (whales, dolphins, porpoises) has, for a long time, attracted the attention of evolutionary biologists. Here we examine cetacean phylogenetic relationships on the basis of analyses of complete mitochondrial genomes that represent all extant cetacean families. The results suggest that the ancestors of recent cetaceans had an explosive evolutionary radiation 30–35 million years before present. During this period, extant cetaceans divided into the two primary groups, Mysticeti (baleen whales) and Odontoceti (toothed whales). Soon after this basal split, the Odontoceti diverged into the four extant lineages, sperm whales, beaked whales, Indian river dolphins and delphinoids (iniid river dolphins, narwhals/belugas, porpoises and true dolphins). The current data set has allowed test of two recent morphological hypotheses on cetacean origin. One of these hypotheses posits that Artiodactyla and Cetacea originated from the extinct group Mesonychia, and the other that Mesonychia/Cetacea constitutes a sister group to Artiodactyla. The current results are inconsistent with both these hypotheses. The findings suggest that the claimed morphological similarities between Mesonychia and Cetacea are the result of evolutionary convergence rather than common ancestry.

The order Cetacea traditionally includes three suborders: the extinct Archaeoceti and the recent Odontoceti and Mysticeti. It is commonly believed that the evolution of ancestral cetaceans from terrestrial to marine (aquatic) life was accompanied by a fast and radical morphological adaptation. Such a scenario may explain why it was, for a long time, difficult to morphologically establish the position of Cetacea in the mammalian tree and even to settle whether Cetacea constituted a monophyletic group.

Biochemical analyses in the 1950s  and 1960s had shown a closer relationship between cetaceans and artiodactyls (even-toed hoofed mammals) than between cetaceans and any other eutherian order and karyological studies in the late 1960s and early 1970s unequivocally supported cetacean monophyly (Arnason, 1969, 1974). The nature of the relationship between cetaceans and artiodactyls was resolved in phylogenetic studies of mitochondrial (mt) cytochrome b (cytb) genes (Irwin and Arnason, 1994; Arnason and Gullberg, 1996) that placed Cetacea within the order Artiodactyla itself as the sister group of the Hippopotamidae (see also Sarich, 1993). The Hippopotamidae/ Cetacea relationship was subsequently supported in studies of nuclear data (Gatesy et al., 1996; Gatesy, 1997) and statistically established in analysis of complete mt genomes (Ursing and Arnason, 1998). The relationship has also been confirmed in analyses of combined nuclear and mt sequences (Gatesy et al., 1999; Cassens et al., 2000) and in studies of short interspersed repetitive elements (SINEs). Artiodactyla and Cetacea are now commonly referred to as Cetartiodactyla.

Previous analyses of the complete cytb gene of more than 30 cetacean species (Arnason and Gullberg, 1996) identified five primary lineages of recent cetaceans, viz., Mysticeti and the four odontocete lineages Physeteridae (sperm whales), Platanistidae (Indian river dolphins), Ziphiidae (beaked whales) and Delphinoidea (iniid river dolphins, porpoises, narwhals and dolphins). However, these studies left unresolved the relationships of the five lineages as well as those between the three delphinoid families Monodontidae (narwhals, belugas), Phocoenidae (porpoises) and Delphinidae (dolphins). Similarly, the relationships between the four mysticete families Balaenidae (right whales), Neobalaenidae (pygmy right whales), Eschrichtiidae (gray whales) and Balaenopteridae (rorquals) were not conclusively resolved in analyses of cytb genes.

Fig. (not shown). Cetartiodactyl relationships and the estimated times of their divergences. The tree was established on the basis of maximum likelihood analysis of the concatenated amino acid (aa) sequences of 12 mt protein-coding genes. Length of alignment 3610 aa. Support values for branches A–H are shown in the insert.
Cetruminantia (branch A) receives moderate support and Cetancodonta (B) strong support. Cetacea (C) splits into monophyletic Mysticeti (baleen whales) and monophyletic Odontoceti (toothed whales). Odontoceti has four basal lineages, Physeteridae (sperm whales: represented by the sperm and pygmy sperm whales), Ziphiidae (beaked whales: bottlenose and Baird’s beaked whales), Platanistidae (Indian river dolphins: Indian river dolphin) and Delphinoidea. Delphinoidea encompasses the families Iniidae (iniid river dolphins: Amazon river dolphin, La Plata dolphin), Monodontidae (narwhals/belugas: narwhal), Phocoenidae (porpoises: harbour porpoise) and Delphinidae (dolphins: white-beaked dolphin). The common odontocete branch and the branches separating the four cetacean lineages are short. These relationships are therefore somewhat unstable (cf. Section 3.1 and Table 1). Iniid river dolphins (F) are solidly nested within the Delphinoidea (E). Thus, traditional river dolphins (Platanistidae + Iniidae) do not form a monophyletic unit. Molecular estimates of divergence times (Sanderson 2002) were based on two calibration points, A/C-60 and O/M-35 (cf. Section 3.2). Due to the short lengths of internal branches, some estimates for these divergences overlap. NJ: neighbor joining; MP: maximum parsimony; LBP: local bootstrap probability; QP: quartet puzzling. The bar shows the number of aa substitutions per site.

The limited molecular resolution among basal cetacean lineages has been known for some time. Studies of hemoglobin and myoglobin (Goodman, 1989; Czelusniak et al., 1990) have either joined Physeteridae and Mysticeti to the exclusion of Delphinoidea (myoglobin data) or Mysticeti and Delphinoidea to the exclusion of Physeteridae (hemoglobin data). Thus, neither of the data sets identified monophyletic Odontoceti by joining the two odontocete lineages (Physeteridae and Delphinoidea) to the exclusion of Mysticeti. A similar instability was recognized and cautioned against in analyses of some mt data, notably, sequences of rRNA genes (Arnason et al., 1993b). The suggestion (Milinkovitch et al., 1993) of a sister group relationship between Physeteridae and the mysticete family Balaenopteridae (rorquals) was based on a myoglobin data set (which joins Physeteridae and Mysticeti to the exclusion of Delphinoidea) that was complemented with partial data of the mt 16S rRNA gene.

The cetancodont divergence times calculated using A/C-60 and O/M-35 as references have been included in Fig. 1. As a result of the short branches separating several cetacean lineages, the estimates of these divergences overlap. The same observation has been made in calculations based on SINE flanking sequences (Nikaido et al., 2001). There is a general consistency between the current and the flanking sequence datings, except for those involving the Balaenopteridae, which are somewhat younger in our analysis than in the SINEs study. The currently estimated age of the divergence between Hippopotamus and Cetacea (c53.5 MYBP) is consistent with the age (>50 MY) of the oldest archaeocete fossils identified so far (Bajpai and Gingerich, 1998). This suggests that the ages allocated to the two references, A/C-60 (the divergence between ruminant artiodactyls and cetancodonts) and O/M-35 (the divergence between odontocetes and mysticetes) are reasonably accurate.

The dating of the divergence between the blue and fin whales is of interest regarding hybridization between closely related mammalian species. Previous molecular analyses (Arnason et al., 1991b; Spilliaert et al., 1991) demonstrated the occurrence of hybridization between these two species. These studies, which were based on three hybrids (one female and two males), showed that either species could be the mother or father in these hybridizations. The two male hybrids had rudimentary testes, whereas the female hybrid was in her second pregnancy. This suggests that the blue and fin whales may be close to the limit for permissible species hybridization among mammals.

The current data set has allowed examination of the coherence between the molecular results and two prevalent morphological hypotheses related to cetacean evolution. The first hypothesis, which in essence originates from Van Valen (1966, 1968), postulates that monophyletic Artiodactyla and monophyletic Cetacea evolved separately from the extinct Palaeocene group Mesonychia. This hypothesis was recently reinforced in a morphological study (Thewissen et al., 2001) that included mesonychians, two archaeocete taxa (Ambuloocetus and Pakicetus) and some extant and fossil artiodactyls. The study of Thewissen et al. (2001) showed a sister group relationship between monophyletic Artiodactyla and monophyletic Cetacea, with Mesonychia as the basal sister group of Artiodactyla/Cetacea, a conclusion consistent with the palaeontological age of Mesonychia relative to that of Artiodactyla and Cetacea. The second hypothesis favours a sister group relationship between Mesonychia and Cetacea with the Mesonychia/Cetacea clade as the sister group of monophyletic Artiodactyla (O’Leary and Geisler, 1999; see also Gatesy and O’Leary, 2001).

Although the position of Mesonychia differs in the two morphological hypotheses, both correspond to a sister group relationship between Cetacea and monophyletic Artiodactyla among extant cetartiodactyls. Thus, both hypotheses can be tested against the current data set. The result of such a test has been included in Table 1, topology (m)(not shown). As evident, both these morphological hypotheses are incongruent with the mitogenomic findings.

Morphological studies have not provided an answer to the question whether mysticetes and odontocetes had separate origins among the archaeocetes (Fordyce and de Muizon, 2001). However, the long common cetacean branch and the short branches separating the five extant cetacean lineages strongly suggest an origin of modern cetaceans from the same archaeocete group (probably the Dorudontidae).

The limbs of Ambulocetus constitute somewhat of an evolutionary enigma. As evident in Thewissen et al.’s (1994) paper, Ambulocetus has very large hind limbs compared to its forelimbs, a difference that is less pronounced in later silhouette drawings of the animal. It is nevertheless evident that evolution from the powerful hindlimbs of Ambulocetus to their rudimentation in archaeocetes constitutes a remarkable morphological reversal if Ambulocetus is connected to the cetacean branch after the separation of the hippopotamid and cetacean lineages.

For natural reasons, systematic schemes have traditionally been based on external morphological characteristics. The rates of morphological and molecular evolution are rarely (if ever) strictly correlated, however, and this may give rise to inconsistency between traditional systematics and molecular findings. The emerging consensus that the order Cetacea resides within another traditional order, Artiodactyla, makes apparent the incongruity in cetartiodactyl nomenclature (Graur and Higgins, 1994). In this instance, a possible solution for maintaining reasonable consistency between nomenclature and phylogeny would be to recognize Cetartiodactyla as an order with three suborders: Suina, Tylopoda and Cetruminantia. According to such a scheme, Cetacea would (together with the Hippopotamidae) constitute a parvorder within the infraorder Cetancodonta.

Cytochrome b and Bayesian inference of whale phylogeny

Laura May-Collado, Ingi Agnarsson
Molecular Phylogenetics and Evolution 38 (2006) 344–354
http://dx.doi.org//10.1016/j.ympev.2005.09.019

In the mid 1990s cytochrome b and other mitochondrial DNA data reinvigorated cetacean phylogenetics by proposing many novel

and provocative hypotheses of cetacean relationships. These results sparked a revision and reanalysis of morphological datasets, and the collection of new nuclear DNA data from numerous loci. Some of the most controversial mitochondrial hypotheses have now become benchmark clades, corroborated with nuclear DNA and morphological data; others have been resolved in favor of more traditional views. That major conflicts in cetacean phylogeny are disappearing is encouraging. However, most recent papers aim specifically to resolve higher-level conflicts by adding characters, at the cost of densely sampling taxa to resolve lower-level relationships. No molecular study to date has included more than 33 cetaceans. More detailed molecular phylogenies will provide better tools for evolutionary studies. Until more genes are available for a high number of taxa, can we rely on readily available single gene mitochondrial data? Here, we estimate the phylogeny of 66 cetacean taxa and 24 outgroups based on Cytb sequences. We judge the reliability of our phylogeny based on the recovery of several deep-level benchmark clades. A Bayesian phylogenetic analysis recovered all benchmark clades and for the Wrst time supported Odontoceti monophyly based exclusively on analysis of a single mitochondrial gene. The results recover the monophyly, with the exception of only one taxa within Cetacea, and the most recently proposed super- and subfamilies. In contrast, parsimony never recovered all benchmark clades and was sensitive to a priori weighting decisions. These results provide the most detailed phylogeny of Cetacea to date and highlight the utility of both Bayesian methodology in general, and of Cytb in cetacean phylogenetics. They furthermore suggest that dense taxon sampling, like dense character sampling, can overcome problems in phylogenetic reconstruction.

Some long standing debates are all but resolved: our understanding of deeper level cetacean phylogeny has grown strong. However, the strong focus of most recent studies, aiming specifically to resolve these higher level conflicts by adding mostly characters rather than taxa, has left our understanding of lower level relationships among whale species lagging behind. Mitogenomic data, for example, is available only for 16 cetacean species, and no molecular study to date has included more than 33 cetaceans. It seems timely to focus on more detailed (genus, and species level) molecular phylogenies. These will provide better tools for detailed evolutionary studies, and are necessary to test existing morphological phylogenetic hypotheses, and current cetacean classification.

We judge the reliability of our phylogeny based on the recovery of the previously mentioned benchmark clades, in addition to the less controversial clades Perissodactyla, Euungulata (sensu Waddell et al., 2001; Perissodactyla+ Cetartiodactyla), Cetacea, and Mysticeti. Because Cytb is thought to be most reliable at lower taxonomic levels (due to high substitution rates), recovering ‘known’ deeper clades gives credibility to these new findings which have not been addressed by studies using few taxa. We compare the performance of Bayesian analyses versus parsimony under four different models, and briefly examine the sensitivity of the results to taxon sampling. We use our results to discuss agreement and remaining conflict in cetacean phylogenetics, and provide comments on current classification.

The Bayesian analysis recovered all seven benchmark clades. Support for five of the benchmark clades is high (100 posterior probabilities) but rather low for Cetancodonta (79) and marginal for the monophyly of Odontoceti. The analysis also recovered all but one family level, and most sub- and superfamily level cetacean taxa. The results broadly corroborate current cetacean classiffcation, while also pointing to some lower-level groups that may need redefinition.

Many recent cetacean phylogenetic studies include relatively few taxa, in part due to a focus on generating more characters to resolve higher level phylogenetics. While addressing crucial questions and providing the backbone for lower level phylogenies, such studies have limited utility for classification, and for comparative evolutionary studies. In some cases sparse taxon sampling may also confound the results. Of course, taxon sampling is usually simply constrained by the availability of character data, but for some reason many studies have opted to include only one, or a few outgroup taxa, even if many are available.

We find that as long as outgroup taxon sampling was extensive, Bayesian analyses of Cytb recovered all the a priori identified benchmark clades. When only a few outgroups were chosen, however, the Bayesian analysis negated Odontoceti monophyly, as have many previous parsimony analyses of mitochondrial DNA. Furthermore, in almost every detailed comparison possible our results mirror the findings O’Leary et al. (2004), the most ‘character-complete’ (but including relatively few cetacean taxa) analysis to date (37,000 characters from morphology, SINE, and 51 gene fragments). This result gives credibility to our findings, including previously untested lower level clades.

  • Monophyly and placement of Mysticeti (baleen whales).
  • Monophyly of Odontoceti (toothed whales)
  • Delphinoids
  • River Dolphins
  • Beaked and sperm whales

A major goal of phylogenetics is a phylogeny of life (i.e., many taxa), based on multiple lines of evidence (many characters of many types). However, when phylogenies based on relatively few characters can be judged reliable based on external evidence (taxonomic congruence with other phylogenies using many characters, but few taxa), they seem like very promising and useful ‘first guess’ hypotheses. The evolution of sexual dimorphism, echolocation, social behavior, and whistles and other communicative signals, and major ecological shifts (e.g., transition to fresh water) are among the numerous interesting questions in cetacean biology that this phylogeny can help answer.

Deep-diving sea lions exhibit extreme bradycardia in long duration dives

Birgitte I. McDonald1, and Paul J. Ponganis
The Journal of Experimental Biology (2014) 217, 1525-1534 http://dx.doi.org:/10.1242/jeb.098558

Heart rate and peripheral blood flow distribution are the primary determinants of the rate and pattern of oxygen store utilization and ultimately breath-hold duration in marine endotherms. Despite this, little is known about how otariids (sea lions and fur seals) regulate heart rate (fH) while diving. We investigated dive fH in five adult female California sea lions (Zalophus californianus) during foraging trips by instrumenting them with digital electrocardiogram (ECG) loggers and time depth recorders. In all dives, dive fH (number of beats/duration; 50±9 beats min−1) decreased compared with surface rates (113±5 beats min−1), with all dives exhibiting an instantaneous fH below resting (<54 beats min−1) at some point during the dive. Both dive fH and minimum instantaneous fH significantly decreased with increasing dive duration. Typical instantaneous fH profiles of deep dives (>100 m) consisted of:

(1) an initial rapid decline in fH resulting in the lowest instantaneous fH of the dive at the end of descent, often below 10 beats min−1 in dives longer than 6 min in duration;
(2) a slight increase in fH to ~10–40 beats min−1 during the bottom portion of the dive; and
(3) a gradual increase in fH during ascent with a rapid increase prior to surfacing.

Thus, fH regulation in deep-diving sea lions is not simply a progressive bradycardia. Extreme bradycardia and the presumed associated reductions in pulmonary and peripheral blood flow during late descent of deep dives should

(a) contribute to preservation of the lung oxygen store,
(b) increase dependence of muscle on the myoglobin-bound oxygen store,
(c) conserve the blood oxygen store and
(d) help limit the absorption of nitrogen at depth.

This fH profile during deep dives of sea lions may be characteristic of deep-diving marine endotherms that dive on inspiration as similar fH profiles have been recently documented in the emperor penguin, another deep diver that dives on inspiration.

The resting ƒH measured in this study (54±6 beats min−1) was lower than predicted for an animal of similar size (~80 beats min−1 for an 80 kg mammal). In part, this may be due to the fact that the sea lions were probably sleeping. The resting ƒH in our study was also lower than previous measurements in captive juvenile California sea lions (87±17 beats min−1, average mass 30 kg)  and wild Antarctic fur seals (78±5 beats min−1, body mass 30–50 kg). However, we found a significant negative relationship between mass and resting ƒH even with our small sample size of five sea lions (resting ƒH = –0.58 Mb +100.26, r2=0.81, F1,3=12.37, P=0.039). For a 30 kg sea lion, this equation predicts a resting ƒH of 83 beats min−1, which is similar to what was measured previously in juvenile sea lions, suggesting this equation may be useful in estimating resting ƒH in sea lions.

The sea lions exhibited a distinct sinus arrhythmia fluctuating between a minimum of 42±9 and a maximum of 87±12 beats min−1, comparable to the sinus arrhythmias described in other diving birds and mammals, including sea lions. The minimum instantaneous ƒH during the sinus arrhythmia was similar to the mean minimum ƒH in dives less than 3 min (37±7 beats min−1), indicating that in dives less than 3 min (estimated cADL), ƒH only decreased to levels observed during exhalation at rest. This is consistent with observations in emperor penguins and elephant seals, where it was proposed that in dives shorter than the aerobic dive limit (ADL) the reduction in ƒH is regulated by a mechanism of cardiorespiratory control similar to that governing the respiratory sinus arrhythmia, with a further reduction only occurring in dives longer than the ADL.

Fig. 3. (not shown) Instantaneous fH and dive depth profiles of a California sea lion (CSL12_2). Data are from (A) a short, shallow dive (1.3 min, 45 m), (B) a mid-duration dive (4.8 min, 239 m) and (C) a long-duration dive (8.5 min, 305 m). Minimum instantaneous fH reached 37 beats min−1 in the short dive
(A) 19 beats min−1 in the mid-duration dive
(B) and 7 beats min−1 in the long duration dive
(C) Prominent features typical of mid- and long-duration dives include

  • a surface interval tachycardia (pre- and post-dive);
  • a steady rapid decrease in fH during initial descent;
  • a gradual decline in fH towards the end of descent with the lowest fH of the dive at the end of descent;
  • a slight increase and sometimes variable fH during the bottom portion of the dive; and
  • a slow increase in fH during ascent,
  • often ending in a rapid increase just before surfacing.

We obtained the first diving ƒH data from wild sea lions on natural foraging trips, demonstrating how they regulate ƒH over a range of dive durations. Sea lions always decreased dive ƒH from surface ƒH values; however, individual sea lions exhibited different dive ƒH, accounting for a significant amount of the variation in the relationship between dive duration and ƒH (intra-individual correlation: 75–81%)). The individual differences in dive ƒH exhibited in this study suggest that different dive capacities of individual sea lions may partially account for the range of dive strategies exhibited in a previous study (Villegas-Amtmann et al., 2011). Despite the individual differences in ƒH, the pattern of the dive ƒH response was similar in all the sea lions. As predicted, sea lions only consistently displayed a true bradycardia on mid- to long- duration dives (>4 min) (Fig. 5A). Additionally, as seen in freely diving phocids, dive ƒH and minimum ƒH were negatively related to dive duration, with the longest duration dives having the lowest dive ƒH and displaying the most intense bradycardia, often below 10 beats min−1 (Fig. 5A,B).

Profiles of mean fH at 10 s intervals of dives

Profiles of mean fH at 10 s intervals of dives

Fig 4.  Profiles of mean fH at 10 s intervals of dives for (A) six duration categories and (B) five depth categories. Standard error bars are shown. Data were pooled from 461 dives performed by five sea lions. The number of dives in each category and the number of sea lions performing the dives in each category are provided in the keys.

The mild bradycardia and the dive ƒH profiles observed in the shorter duration dives (<3 min) were similar to those observed in trained juvenile California sea lions and adult Stellar sea lions, but much more intense than ƒH observed in freely diving Antarctic fur seals. Surprisingly, although dive ƒH of trained Steller sea lions was similar, Steller sea lions regularly exhibited lower minimum ƒH, with minimum ƒH almost always less than 20 beats min−1 in dives less than 2 min in duration. In the wild, California sea lions rarely exhibited a minimum ƒH less than 20 beats min−1 in similar duration dives (Fig. 5B), suggesting greater blood oxygen transport during these natural short-duration dives.

Fig. 5. (not shown)  fH decreases with increasing dive duration. Dive duration versus (A) dive fH (total number of beats/dive duration), (B) minimum instantaneous fH and (C) bottom fH (total beats at bottom of dive/bottom time) for California sea lions (461 dives from five sea lions).

Although California sea lions are not usually considered exceptional divers, they exhibited extreme bradycardia, comparable to that of the best diving phocids, during their deep dives. In dives greater than 6 min in duration, minimum ƒH was usually less than 10 beats min−1 and sometimes as low as 6 beats mins−1 (Fig. 5B), which is similar to extreme divers such as emperor penguins (3 beats min−1), elephant seals (3 beats min−1), grey seals (2 beats min−1) and Weddell seals (<10 beats min−1), and even as low as what was observed in forced submersion studies. Thus, similar to phocids, the extreme bradycardia exhibited during forced submersions is also a routine component of the sea lion’s physiological repertoire, allowing them to perform long-duration dives.

While the degree of bradycardia observed in long dives of California sea lions was similar to the extreme bradycardia observed in phocids, the ƒH profiles were quite different. In general, phocid ƒH decreases abruptly upon submergence. The intensity of the initial phocid bradycardia either remains relatively stable or intensifies as the dive progresses, and does not start to increase until the seal begins its ascent. In contrast, the ƒH profiles of sea lions were more complex, showing a more gradual decrease during descent, with the minimum ƒH of the dive usually towards the end of descent (Figs 3, 6). There was often a slight increase in ƒH during the bottom portion of the dive, and as soon as the sea lions started to ascend, the ƒH slowly started to increase, often becoming irregular during the middle of ascent, before increasing rapidly as the sea lion approached the surface.

Fig. 6. (not shown) Instantaneous fH and dive depth profiles of the longest dive (10.0 min, 385 m) from a California sea lion (CSL12_1). During this dive, instantaneous fH reached 7 beats min−1 and was less than 20 beats min−1 for over 5.5 min. Post-dive fH was high in the first 0.5–1 min after surfacing, but then declined to ~100 beats min−1 towards the end of the surface interval.

Implications for pulmonary gas exchange

The moderate dive ƒH in short, shallow dives compared with the much slower ƒH of deep long-duration dives suggests more pulmonary blood flow and greater potential for reliance on lung O2. Most of these dives were to depths of less than 100 m (well below the estimated depth of lung collapse near 200 m), so maintenance of a moderate ƒH during these dives may allow sea lions to maximise use of the potentially significant lung O2 stores (~16% of total body O2 stores) throughout the dive. This is supported by venous blood O2 profiles, where, occasionally, there was no decrease in venous blood O2 between the beginning and end of the dive; this can only occur if pulmonary gas exchange continues throughout the dive. Greater utilization of the lung O2 store in sea lions is consistent with higher dive ƒH in other species that both dive on inspiration and typically perform shallow dives (dolphins, porpoises, some penguin species), and in deeper diving species when they perform shallow dives (emperor penguins).

In deeper dives of sea lions, although ƒH was lower and bradycardia more extreme, the diving ƒH profiles suggest that pulmonary gas exchange is also important. In long-duration dives, even though ƒH started to decrease upon or shortly after submergence, the decrease was not as abrupt as in phocids. Additionally, in long deep dives, despite having overall low dive ƒH, there were more heart beats before resting ƒH was reached compared with short, shallow dives. In dives less than 3 min in duration, there were ~10–15 beats until instantaneous ƒH reached resting values. In longer duration dives (>3 min), there were usually ~30–40 beats before instantaneous ƒH reached resting values. We suggest the greater number of heart beats early in these deeper dives enables more gas exchange and blood O2 uptake at shallow depths, thus allowing utilisation of the postulated larger respiratory O2 stores in deeper dives The less abrupt decline in ƒH we observed in sea lions is similar to the more gradual declines documented in emperor penguins and porpoises, where it has also been proposed that the gradual decrease in ƒH allows them to maximise pulmonary gas exchange at shallower depths. However, as sea lions swam deeper, ƒH decreased further (Figs 3, 6), and by 200 m depth (the approximate depth of lung collapse, instantaneous ƒH was 14 beats min−1. Such an extreme decline in ƒH in conjunction with increased pulmonary shunting due to lung compression at greater depths will result in minimization of both O2 and N2 uptake by blood, even before the depth of full lung collapse (100% pulmonary shunt) is reached.

Implications for blood flow

ƒH is often used as a proxy to estimate blood flow and perfusion during diving because of the relative ease of its measurement. This is based on the assumption that stroke volume does not change during diving in sea lions, and, hence, changes in ƒH directly reflect changes in cardiac output. As breath-hold divers maintain arterial pressure while diving, changes in cardiac output should be associated with changes in peripheral vascular resistance and changes in blood flow to tissues. In Weddell seals, a decrease in cardiac output of ~85% during forced submersions resulted in an 80–100% decrease in tissue perfusion in all tissues excluding the brain, adrenal glands and lung. Sea lions exhibited extremely low instantaneous ƒH values that often remained low for significant portions of the dive (Figs 4, 6), suggesting severe decreases in tissue perfusion in dives greater than 5 min in duration. In almost all dives greater than 6 min in duration, instantaneous ƒH reached 10 beats min−1, and stayed below 20 beats min−1 for more than a minute. At a ƒH of 20 beats min−1, cardiac output will be ~36% of resting cardiac output and only about 18% of average surface cardiac output. At these levels of cardiac suppression, most of this flow should be directed towards the brain and heart.

Conclusions

We successfully obtained diving ƒH profiles from a deep-diving otariid during natural foraging trips. We found that

(1) ƒH decreases during all dives, but true and more intense bradycardia only occurred in longer duration dives and
(2) in the longest duration dives, ƒH and presumed cardiac output were as low as 20% of resting values.

We conclude that, although initial high ƒH promotes gas exchange early in deep dives, the extremely low ƒH in late descent of deep dives (a) preserves lung O2, (b) conserves blood O2, (c) increases the dependence of muscle on myoglobin-bound O2 and (d) limits N2 absorption at depth. This ƒH profile, especially during the late descent/early bottom phase of deep dives is similar to that of deep-diving emperor penguins, and may be characteristic of deep diving endotherms that dive on inspiration.

Dive duration was the fixed effect in all models, and to account for the lack of independence caused by having many dives from the same individual, individual (sea lion ID) was included as a random effect. Covariance and random effect structures of the full models were evaluated using Akaike’s information criterion (AIC) and examination of residual plots. AICs from all the tested models are presented with the best model in bold.

Additionally, dives were classified as short-duration (less than 3 min, minimum cADL), mid-duration (3–5 min, range of cADLs) or long-duration (>5 min) dives. Differences in pre-dive ƒH, dive ƒH, minimum ƒH, post-dive ƒH, and heart beats to resting between the categories were investigated using mixed effects ANOVA, followed by post hoc Tukey tests. In all models, dive duration category was the fixed effect and individual (sea lion ID) was included as a random effect. Model fit was accessed by examination of the residuals. All means are expressed ±s.d. and results of the Tukey tests were considered significant at P<0.05. Statistical analysis was performed in R.

Investigating Annual Diving Behaviour by Hooded Seals (Cystophora cristata) within the Northwest Atlantic Ocean

Julie M. Andersen, Mette Skern-Mauritzen, Lars Boehme
PLoS ONE 8(11): e80438. http://dx.doi.org:/10.1371/journal.pone.0080438

With the exception of relatively brief periods when they reproduce and molt, hooded seals, Cystophora cristata, spend most of the year in the open ocean where they undergo feeding migrations to either recover or prepare for the next fasting period. Valuable insights into habitat use and diving behavior during these periods have been obtained by attaching Satellite Relay Data Loggers (SRDLs) to 51 Northwest (NW) Atlantic hooded seals (33 females and 18 males) during icebound fasting periods (200422008). Using General Additive Models (GAMs) we describe habitat use in terms of First Passage Time (FPT) and analyze how bathymetry, seasonality and FPT influence the hooded seals’ diving behavior described by maximum dive depth, dive duration and surface duration. Adult NW Atlantic hooded seals exhibit a change in diving activity in areas where they spend .20 h by increasing maximum dive depth, dive duration and surface duration, indicating a restricted search behavior. We found that male and female hooded seals are spatially segregated and that diving behavior varies between sexes in relation to habitat properties and seasonality. Migration periods are described by increased dive duration for both sexes with a peak in May, October and January. Males demonstrated an increase in dive depth and dive duration towards May (post-breeding/pre-molt) and August–October (post-molt/pre-breeding) but did not show any pronounced increase in surface duration. Females dived deepest and had the highest surface duration between December and January (post-molt/pre-breeding). Our results suggest that the smaller females may have a greater need to recover from dives than that of the larger males. Horizontal segregation could have evolved as a result of a resource partitioning strategy to avoid sexual competition or that the energy requirements of males and females are different due to different energy expenditure during fasting periods.

Novel locomotor muscle design in extreme deep-diving whales

P. Velten, R. M. Dillaman, S. T. Kinsey, W. A. McLellan and D. A. Pabst
The Journal of Experimental Biology 216, 1862-1871
http://dx.doi.org:/10.1242/jeb.081323

Most marine mammals are hypothesized to routinely dive within their aerobic dive limit (ADL). Mammals that regularly perform deep, long-duration dives have locomotor muscles with elevated myoglobin concentrations that are composed of predominantly large, slow-twitch (Type I) fibers with low mitochondrial volume densities (Vmt). These features contribute to extending ADL by increasing oxygen stores and decreasing metabolic rate. Recent tagging studies, however, have challenged the view that two groups of extreme deep-diving cetaceans dive within their ADLs. Beaked whales (including Ziphius cavirostris and Mesoplodon densirostris) routinely perform the deepest and longest average dives of any air-breathing vertebrate, and short-finned pilot whales (Globicephala macrorhynchus) perform high-speed sprints at depth. We investigated the locomotor muscle morphology and estimated total body oxygen stores of several species within these two groups of cetaceans to determine whether they

(1) shared muscle design features with other deep divers and
(2) performed dives within their calculated ADLs.

Muscle of both cetaceans displayed high myoglobin concentrations and large fibers, as predicted, but novel fiber profiles for diving mammals. Beaked whales possessed a sprinterʼs fiber-type profile, composed of ~80% fast-twitch (Type II) fibers with low Vmt. Approximately one-third of the muscle fibers of short-finned pilot whales were slow-twitch, oxidative, glycolytic fibers, a rare fiber type for any mammal. The muscle morphology of beaked whales likely decreases the energetic cost of diving, while that of short-finned pilot whales supports high activity events. Calculated ADLs indicate that, at low metabolic rates, both beaked and short-finned pilot whales carry sufficient onboard oxygen to aerobically support their dives.

Serial cross-sections of the m. longissimus dorsi of Mesoplodon densirostris

Serial cross-sections of the m. longissimus dorsi of Mesoplodon densirostris

Fig. Serial cross-sections of the m. longissimus dorsi of Mesoplodon densirostris (A–D) and Globicephala macrorhynchus (E–H). Scale bars, 50μm. Muscle sections stained for the alkaline (A,E) and acidic (B,F) preincubations of myosin ATPase were used to distinguish Type I and II fibers. Muscle sections stained for succinate dehydrogenase (C,G) and α-glycerophosphate dehydrogenase (D,H) were used to distinguish glycolytic (gl), oxidative (o) and intermediate (i) fibers.

Previous studies of the locomotor muscles of deep-diving marine mammals have demonstrated that these species share a suite of adaptations that increase onboard oxygen stores while slowing the rate at which these stores are utilized, thus extending ADL. Their locomotor muscles display elevated myoglobin concentrations and are composed predominantly of large Type I fibers. Vmt are also lower in deep divers than in shallow divers or athletic terrestrial species. The results of this study indicate that beaked whales and short-finned pilot whales do not uniformly display these characteristics and that each possesses a novel fiber profile compared with those of other deep divers.

The phylogeny of Cetartiodactyla: The importance of dense taxon sampling, missing data, and the remarkable promise of cytochrome b to provide reliable species-level phylogenies

Ingi Agnarsson, Laura J. May-Collado
Molecular Phylogenetics and Evolution 48 (2008) 964–985
http://dx.doi.org:/10.1016/j.ympev.2008.05.046

We perform Bayesian phylogenetic analyses on cytochrome b sequences from 264 of the 290 extant cetartiodactyl mammals (whales plus even-toed ungulates) and two recently extinct species, the ‘Mouse Goat’ and the ‘Irish Elk’. Previous primary analyses have included only a small portion of the species diversity within Cetartiodactyla, while a complete supertree analysis lacks resolution and branch lengths limiting its utility for comparative studies. The benefits of using a single-gene approach include rapid phylogenetic estimates for a large number of species. However, single-gene phylogenies often differ dramatically from studies involving multiple datasets suggesting that they often are unreliable. However, based on recovery of benchmark clades—clades supported in prior studies based on multiple independent datasets—and recovery of undisputed traditional taxonomic groups, Cytb performs extraordinarily well in resolving cetartiodactyl phylogeny when taxon sampling is dense. Missing data, however, (taxa with partial sequences) can compromise phylogenetic accuracy, suggesting a tradeoff between the benefits of adding taxa and introducing question marks. In the full data, a few species with a short sequences appear misplaced, however, sequence length alone seems a poor predictor of this phenomenon as other taxa.

The mammalian superorder Cetartiodactyla (whales and eventoed ungulates) contains nearly 300 species including many of immense commercial importance (cow, pig, and sheep) and of conservation interest and aesthetic value (antelopes, deer, giraffe, dolphins, and whales) (MacDonald, 2006). Certain members of this superorder count among the best studied organisms on earth, whether speaking morphologically, behaviorally, physiologically or genetically. Understanding the interrelationships among cetartiodactyl species, therefore, is of obvious importance with equally short sequences were not conspicuously misplaced. Although we recommend awaiting a better supported phylogeny based on more character data to reconsider classification and taxonomy within Cetartiodactyla, the new phylogenetic hypotheses provided here represent the currently best available tool for comparative species-level studies within this group. Cytb has been sequenced for a large percentage of mammals and appears to be a reliable phylogenetic marker as long as taxon sampling is dense. Therefore, an opportunity exists now to reconstruct detailed phylogenies of most of the major mammalian clades to rapidly provide much needed tools for species-level comparative studies.

Our results support the following relationship among the four major cetartiodactylan lineages (((Tylopoda ((Cetancodonta (Ruminantia + Suina))), with variable support. This arrangement has not been suggested previously, to our knowledge (see review in O’Leary and Gatesy, 2008 and discussion).

Relationships among clades within Cetancodonta are identical to those found by May-Collado and Agnarsson (2006).

Within Ruminantia all our analyzes suggest the following relationships among families: (((((Tragulidae((((Antilocapridae(((Giraffidae(( Cervidae(Moschidae + Bovidae))))) with relatively high support, supporting the subdivision of Ruminantia into Tragulina and Pecora.
In the rare cases where our results are inconsistent with benchmark clades, ad hoc explanations seem reasonable. The placement of M. meminna (Tragulidae) within Bovidae is likely an artifact of missing data, although remarkably it is the only conspicuous misplacement of a species across the whole phylogeny at the family level (while three species appear to be misplaced at the subfamily level within Cervidae in the full analysis, see Fig. 5a). This is supported by the fact that the placement of Moschiola receives low support, and the removal of Moschiola prior to analysis increases dramatically the support for clades close to where it nested (not shown, analysis available from authors), suggesting it had a tendency to ‘jump around’. Two other possibilities cannot be ruled out, however. One, that possibly the available sequence in Genbank may be mislabeled. And second, it should be kept in mind that the validity of Tragulidae has never been tested with molecular data including more than two species.

Oxygen and carbon dioxide fluctuations in burrows of subterranean blind mole rats indicate tolerance to hypoxic–hypercapnic stresses

Imad Shams, Aaron Avivi, Eviatar Nevo
Comparative Biochemistry and Physiology, Part A 142 (2005) 376 – 382
http://dx.doi.org:/10.1016/j.cbpa.2005.09.003

The composition of oxygen (O2), carbon dioxide (CO2), and soil humidity in the underground burrows from three species of the Israeli subterranean mole rat Spalax ehrenbergi superspecies were studied in their natural habitat. Two geographically close populations of each species from contrasting soil types were probed. Maximal CO2 levels (6.1%) and minimal O2 levels (7.2%) were recorded in northern Israel in the breeding mounds of S. carmeli in a flooded, poor drained field of heavy clay soil with very high volumetric water content. The patterns of gas fluctuations during the measurement period among the different Spalax species studied were similar. The more significant differentiation in gas levels was not among species, but between neighboring populations inhabiting heavy soils or light soils: O2 was lower and CO2 was higher in the heavy soils (clay and basaltic) compared to the relatively light soils (terra rossa and rendzina). The extreme values of gas concentration, which occurred during the rainy season, seemed to fluctuate with partial flooding of the tunnels, animal digging activity, and over-crowded breeding mounds inhabited by a nursing female and her offspring. The gas composition and soil water content in neighboring sites with different soil types indicated large differences in the levels of hypoxic–hypercapnic stress in different populations of the same species. A growing number of genes associated with hypoxic stress have been shown to exhibit structural and functional differences between the subterranean Spalax and the aboveground rat (Rattus norvegicus), probably reflecting the molecular adaptations that Spalax went through during 40 million years of evolution to survive efficiently in the severe fluctuations in gas composition in the underground habitat.

map of the studied sites

map of the studied sites

Schematic map of the studied sites: S. galili (2n =52): 1— Rehania (chalk); 2— Dalton (basaltic); S. golani (2n =54): 3— Majdal Shams (terra tossa); 4—Masa’ada (basaltic soils); S. carmeli (2n =58): 5— Al-Maker (heavy clay); 6— Muhraqa (terra rossa).

Comparison of gas composition (O2 and CO2) and water content between light and heavy soils inhabited by S. carmeli

Comparison of gas composition (O2 and CO2) and water content between light and heavy soils inhabited by S. carmeli

Comparison of gas composition (O2 and CO2) and water content between light and heavy soils inhabited by S. carmeli, Al-Maker (heavy soil) and Muhraqa (light soil). AverageTSD of measurements in the burrows of approximately 10 animals at a given date is presented. **p <0.01, T-test and Mann– Whitney test).

Subterranean mammals, which live in closed underground burrow systems, experience an atmosphere that is different from the atmosphere above-ground. Gas exchange between these two atmospheres depends on diffusion through the soil, which in turn, depends on soil particle size, water content, and burrow depth. Heavy soils (clay and basaltic), hold water and have little air space for gas diffusion. A large deviation from external gas composition is found in the burrows of Spalax living in these soil types. The maximal measured concentration of CO2 was 6.1% in Spalax breeding mounds, which is one of the highest concentrations among studied mammals in natural conditions. At the same time 7.2% O2 was measured in water saturated heavy clay soil

seasonal variation from August to March in mean O2, CO2, and soil water content

seasonal variation from August to March in mean O2, CO2, and soil water content

Example of seasonal variation from August to March in mean O2, CO2, and soil water content (VWC) in the Al-Maker population (2n =58, heavy soil). Values are presented as mean TSD.

In this study new data were presented for a wild mammal that survives in an extreme hypoxic–hypercapnic environment. Interestingly, the very low concentrations of O2 experienced by Spalax are correlated with the expression pattern of hypoxia related genes.  So far, we have shown higher and longer-term mRNA expression of erythropoietin, the main factor that regulates the level of circulating red blood cells, in subterranean Spalax compared to the above-ground rat in response to hypoxic stress, as well as differences in the response of erythropoietin to hypoxia in different populations of Spalax experiencing different hypoxic stress in nature. We also demonstrated that erythropoietin pattern of expression is different in Spalax than in Rattus throughout development, a pattern suggesting more efficient hypoxic tolerance in Spalax starting as early as in the embryonic stages. Furthermore, vascular endothelial growth factor (VEGF), which is a critical angiogenic factor that responds to hypoxia, is constitutively expressed at maximal levels in Spalax muscles, the most energy consuming tissue during digging. This level is 1.6-fold higher than in Rattus muscles and is correlated with significantly higher blood vessel concentration in the Spalax muscles compared to the Rattus muscles. Likewise, myoglobin the globin involved in oxygen homeostasis in skeletal muscles, exhibits different expression pattern under normoxia and in response to hypoxia in Spalax muscles compared to rat muscles as well as between different populations of Spalax exposed to different hypoxic stress in nature (unpublished results). Similarly, neuroglobin, a brain-specific globin involved in reversible oxygen binding, i.e., presumably in cellular homeostasis, is expressed differently in the Spalax brain compared to Rattus brain. Like erythropoietin and myoglobin also neuroglobin is expressed differently in Spalax populations experiencing different oxygen supply (unpublished results). Furthermore, Spalax p53 harbors two amino acid substitutions in its binding domain, which are identical to mutations found in p53 of human cancer cells. These substitutions endow Spalax p53 with several-fold higher activation of cell arrest and DNA repair genes compared to human p53 and favor activation of DNA repair genes over apoptotic genes. The study of specific tumoral variants indicates that such preference of growth arrest over apoptosis possibly results as a response to the hypoxic environmental stress known in tumors. Differences in the structure of other molecules related to homeostasis, namely, hemoglobin, haptoglobin (Nevo, 1999), and cytoglobin (unpublished) were also observed in Spalax.

Stress, adaptation, and speciation in the evolution of the blind mole rat, Spalax, in Israel

Eviatar Nevo
Molecular Phylogenetics and Evolution 66 (2013) 515–525
http://dx.doi.org/10.1016/j.ympev.2012.09.008

Environmental stress played a major role in the evolution of the blind mole rat superspecies Spalax ehrenbergi, affecting its adaptive evolution and ecological speciation underground. Spalax is safeguarded all of its life underground from aboveground climatic fluctuations and predators. However, it encounters multiple stresses in its underground burrows including darkness, energetics, hypoxia, hypercapnia, food scarcity, and pathogenicity. Consequently, it evolved adaptive genomic, proteomic, and phenomic complexes to cope with those stresses. Here I describe some of these adaptive complexes, and their theoretical and applied perspectives. Spalax mosaic molecular and organismal evolution involves reductions or regressions coupled with expansions or progressions caused by evolutionary tinkering and natural genetic engineering. Speciation of Spalax in Israel occurred in the Pleistocene, during the last 2.00–2.35 Mya, generating four species associated intimately with four climatic regimes with increasing aridity stress southwards and eastwards representing an ecological speciational adaptive trend: (Spalax golani, 2n = 54?S. galili, 2n = 52?S. carmeli, 2n = 58?S. judaei, 2n = 60). Darwinian ecological speciation occurred gradually with relatively little genetic change by Robertsonian chromosomal and genic mutations. Spalax genome sequencing has just been completed. It involves multiple adaptive complexes to life underground and is an evolutionary model to a few hundred underground mammals. It involves great promise in the future for medicine, space flight, and deep-sea diving.

Stress is a major driving force of evolution (Parsons, 2005; Nevo, 2011). Parsons defined stress as the ‘‘environmental factor causing potential injurious changes to biological systems with a potential for impacts on evolutionary processes’’. The global climatic transition from the middle Eocene to the early Oligocene (45–35 Ma = Million years ago) led to extensive convergent evolution underground of small subterranean mammals across the planet (Nevo, 1999; Lacey et al., 2000; Bennett and Faulkes, 2000; Begall et al., 2007). The subterranean ecotope provided small mammals with shelter from predators and extreme aboveground climatic stressful fluctuations of temperature and humidity. However, they had to evolve genomic adaptive complexes for the immense underground stresses of darkness, energy for burrowing in solid soil, low productivity and food scarcity, hypoxia, hypercapnia, and high infectivity. These stresses have been described in Nevo (1999, 2011) and Nevo et al. (2001); and Nevo list of Spalax publication at http://evolution.haifa.ac.il with many cited references relevant to these stresses).

blind subterranean mole rat of the Spalax ehrenbergi superspecies

blind subterranean mole rat of the Spalax ehrenbergi superspecies

The blind subterranean mole rat of the Spalax ehrenbergi superspecies in Israel. An extreme example of adaptation to life underground

Circadian rhythm and genes

adaptive circadian genes. We identified the circadian rhythm of Spalax
(Nevo et al., 1982) and described, cloned, sequenced, and expressed several circadian genes in Spalax. These include Clock, MOP3, three Period (Per), and cryptochromes (Avivi et al., 2001, 2002, 2003). The Spalax circadian genes are differentially conserved, yet characterized by a significant number of amino acid substitutions. The glutamine-rich area of Clock, which is assumed to function in circadian rhythmicity, is expanded in Spalax compared with that of mice and humans and is different in amino acid composition from that of rats. All three Per genes of Spalax oscillate with a periodicity of 24 h in the suprachaismatic nucleus, eye, and Harderian gland and are expressed in peripheral organs. Per genes are involved in clock resetting. Spalax Per 3 is unique in mammals though its function is still unresolved. The Spalax Per genes contribute to the unique adaptive circadian rhythm to life underground. The cryptochrome (Cry) genes, found in animals and plants, act both as photoreceptors and as ingredients of the negative feedback mechanism of the biological Clock. The CRY 1 protein is significantly closer to the human homolog than to that of mice, as was also shown in parts of the immunogenetic system. Both Cry 1 and Cry 2 mRNAs were found in the SCN, eye, harderian gland, and in peripheral tissues. Remarkably, the distinctly hypertrophied harderian gland is central in Spalax’s unique underground circadian rhythmicity (Pevet et al., 1984).

  • Spalax eye mosaic evolution
  • Gene expression in the eye of Spalax
  • Brain evolution in Spalax to underground stresses
  • Spalax: four species in Israel

The morphological, physiological, and behavioral Spalax eye patterns are underlain by gene expression representing regressive and progressive associated transcripts. Regressive transcripts involve B-2 microglobulin, transketolase, four keratins, alpha enolase, and different heat shock proteins. Several proteins may be involved in eye degeneration. These include heat shock protein 90alpha (hsp90alpha), found also in the blind fish Astyanax mexicanus, two transcripts of programmed cell death proteins, oculospanin, and peripherin 2, both belonging to the Tetraspanin family, in which 60 different mutations cause eye degeneration in humans. Several progressive transcripts in the Spalax eye are found in the retina of many mammals involving gluthatione, peroxidase 4, B spectrin, and Ankyrin; the last two characterize rod cells in the retina. Some transcripts are involved in metabolic processing of retinal, a vertebrate key component in phototransduction, and a relative of vitamin A.

cross section of the developing eye of the mole rat

cross section of the developing eye of the mole rat

Light micrographs showing cross section of the developing eye of the mole rat Spalax ehrenbergi. (A) Optic cup and lens vesicle initially develop normally (x100). (B) Eye at a later embryonic stage. Note appearance of iris-ciliary body rudiment (arrows), and development of the lens nucleus (L). ON, optic nerve (x100). (C) Eye at a still later fetal stage. Note massive growth of the iris-ciliary body complex colobomatous opening (arrow) (x100). (D) Early postnatal stage. The iris-ciliary body complex completely fills the chamber. The lens is vascularized and vacuolated (x100). (E) Adult eye. Eyelids are completely closed and pupil is absent. Note atrophic appearance of the optic disc region (arrow) (x65). (F) Higher magnification of the adult retina. The different retinal layers are retained: PE, pigment epithelium: RE, receptor layer; ON, outer nuclear layer: IN, inner nuclear layer; GC, ganglion cell layer (x500) (from Sanyal et al., 1990, Fig. 1).

The brains of subterranean mammals underwent dramatic evolution in accordance with underground stresses for digging and photoperiodic perception associated with vibrational, tactile, vocal, olfactory, and magnetic communication systems replacing sight, as is seen in Spalax. The brain of Spalax is twice as large as that of the laboratory rat of the same body size. The somatosensory region in the isocortex of Spalax is 1.7 times, the thalamic nuclei 1.3 times, and the motor cortex 3.1 times larger than in the sighted laboratory rat Rattus norvegicus matched to body size.

The ecological stress determinant in Spalax brain evolution is highlighted by the four species of the Spalax ehrenbergi superspecies in Israel. They differentiated chromosomally (by means of Robertsonian mutations and fission), allopatrically, and clinally southwards into four species associated with different climatic regimes, following the gradient of increasing aridity stress and decreasing predictability southwards towards the desert: Spalax galili (2n = 52) ->S. golani (2n = 54)->S. carmeli (2n = 58)->S. judaei (2n = 60), and eastwards S. galili ->S. golani (2n = 52–>54) (Fig. 2). This chromosomal speciation trend southwards is associated with the regional aridity stress southwards (and eastwards) in Israel, budding new species adapted genomically, proteomically, and phenomically (i.e., in morphology, physiology, and behavior) to increasing stresses of higher solar radiation, temperature, and drought southwards (Nevo, 1999; Nevo et al., 2001; Nevo
list of Spalax at http://evolution.haifa.ac.il). A uniquely recent discovery of incipient sympatric ecological speciation at a microscale in Spalax triggered by local stresses occurs within Spalax galili.

retinal input to primary visual structures in Spalax

retinal input to primary visual structures in Spalax

Relative degree of retinal input to primary visual structures in Spalax, hamster, rat, and Spalacopus cyanus (South American Octodontidae, ‘‘coruro’’). These rodents are of similar body size (120–140 g). B. Relative degree of change in the proportions of retinal input to different primary visual structures in Spalax compared with measures obtained in other rodents. A relative progressive development in Spalax is seen in structures involved in photoperiodic and neuroendocrine functions (SCN, BNST).The main regressive feature is the drastic relative reduction of retinal input to the superior colliculus. The main regressive feature is the drastic reduction of retinal input to the superior colliculus. The relative size of other visual structures in Spalax is modified compared to that of the other species. c. Comparison of the absolute size (volume, mm3 x 10-4) of visual structures in Spalax and other rodents. The size of the SCN is equivalent in all species. The vLGN and dLGN are reduced by 87–93% in Spalax. The retino-recipient layers of the superior colliculus are reduced by 97%. Abbreviations: SCN: suprachiasmatic nucleus; BNST: bed nucleus of the stria terminalis; dLGN: dorsal lateral geniculate nucleus; SC: superior colliculus [From Cooper et al., 1993 (Fig 3)].

Subterranean life has a high energetic cost if an animal has to burrow in order to obtain its food. For a 150 g Thomomys bottae, burrowing 1 m may be 360–3400 times more expensive energetically than moving the same distance on the surface (Vleck, 1979). Mean rates of oxygen consumption during burrowing at 22 oC are from 2.8 to 7.1 times the RMR. Vleck developed a model examining the energetics of foraging by burrowing and found that, in the desert, Thomomys adjusts the burrow segment length to minimize the cost of burrowing. Since burrowing becomes less economic as body size increases, Vleck (1981) predicted that the maximum possible body size that a subterranean mammal can attain depends on a balance between habitat productivity and the cost of burrowing in local soils. Vleck’s cost of burrowing hypothesis has been verified in multiple cases. Heth (1989) demonstrated longer burrows in the rendzina soil and shorter ones in the terra rossa soil, associating lower productivity in the former for Spalax.

Food is a limiting factor for subterranean mammals. The abundance and distribution of food explain some of the ecological, physiological, and behavioral characteristics of subterranean mammals. In a field test of Spalax foraging strategy, we concluded that Spalax was a generalist due to the constraints of the subterranean ecotope. Restricted foraging time primarily during the winter when soil is wet, and the high energetic investment of tunneling to get to food items is significantly reduced than in summertime.
We also identified a decrease in the basic metabolic rate towards the desert, i.e., economizing energetics. The maintenance of adequate O2 transport in a subterranean mammal confronting hypoxia requires adaptation along the O2 transport system, achieved by increasing the flow of O2 in the convection systems (ventilation and perfusion) and by reduction of oxygen pressure (PO2) gradients at the diffusion barriers (lung blood, blood-tissue (Arieli, 1990). The PO2 gradient between blood capillaries and respiring mitochondria capillaries is large, and any adaptation at this level could be significant for O2 transport. Reduction of diffusion distance in a muscle can be achieved, like in Spalax, by increasing the number of capillaries that surround muscle fiber or by reducing fiber areas.

Geographic distribution in Israel of the four chromosomal species belonging to the S. ehrenbergi superspecies

Geographic distribution in Israel of the four chromosomal species belonging to the S. ehrenbergi superspecies

Geographic distribution in Israel of the four chromosomal species belonging to the S. ehrenbergi superspecies that are separated by narrow hybrid zones (2n = 52, 54, 58, and 60, now named as S. galili, S. golani, S. carmeli, and S. judaei, respectively; see Nevo et al., 2001).

Spalacid evolution, based on mtDNA, is driven by climatic oscillations and stresses. The underground ecotope provided subterranean mammals with shelter from extreme climate (temperature and humidity) fluctuations, and predators. However, they had to extensively and intensively adapt to the multiple underground stresses (darkness, energetic, low productivity and
food scarcity, hypoxia, hypercapnia, and high infectivity). All subterranean mammals, including spalacids as an extreme case, share convergent molecular and organismal adaptations to their shared unique underground ecotope. Evolution underground, as exemplified here in spalacids, led to mosaic molecular and organismal evolutionary syndromes to cope with multiple stresses.

Speciation involves all rates – from gradual to rapid. Subterranean mammals, with the spalacid example discussed above, provide uniquely rich evolutionary global tests of speciation and adaptation, convergence, regression, progression, and mosaic evolutionary processes. Adaptation and speciation underground was one of the most dramatic natural experiments verifying Darwinian evolution.

The Spalax genome sequencing has just been completed. It is being analyzed and will soon be published in 2012. This will be a milestone in understanding how numerous mammals across the globe, who found underground shelter from climatic fluctuations and stresses above ground, cope with the new suite of stresses they encountered underground, demanding a new engineering overhaul on all organizational levels, selecting for adaptive complexes to cope with the new underground stresses. The main current and future challenges are to compare and contrast genome sequences and identify the genomic basis of adaptation and speciation.

This global Cenozoic experiment could answer the following open questions: How heterozygous is the whole genome? How prevalent are retrotransposons and what is their functional role? How many genes are involved in the Spalax genome and how are they regulated? What are the genic and regulatory networks resisting the multiple stresses underground? How much of the Spalax genome is conserved and how much is reorganized to cope with the underground stresses? How is the solitary blind mole rat, Spalax, different from the social naked mole rat Heterocephalus? How are the processes of reduction, expansion, and genetic tinkering and engineering reflected across the genome? How effective is copy number variation in regulation? Is there similarity in the transcriptomes of subterranean mammals? How could we harness the rich genome repertoire of Spalax to revolutionize medicine, especially in the realm of hypoxia tolerance and the related major diseases of the western world, e.g., cancer, stroke, and cardiovascular diseases? What is the phylogenetic origin of Spalax? How much of the Spalax genome represents its phylogenetic roots and how much of coding and noncoding genomic regions are shared with other subterranean mammals across the globe in adapting to life underground?

The Atmospheric Environment of the Fossorial Mole Rat (Spalax Ehrenbergi): Effects of Season, Soil Texture, Rain, Temperature and Activity

  1. Arieli
    Comp Biochen Physiol. 1978; 63A:569-5151. The fossorial mole rat (Spalax ehrenbergi) may inhabit heavy soil with low gas permeability.
  2. Air composition in burrows in heavy soil deviates from atmospheric air more than that of burrows in light soil.
  3. In winter and spring O2 and CO2 concentrations in breeding mounds were 16.5% O2 and 2.5-3x CO2 and the extreme values measured were 14.0% O2 and 4.8% Cot.
  4. Hypoxia and hypercapnia in the burrow develop shortly after rain and when ambient temperature drops.
  5. Composition of the burrows air is influenced by the solubility of CO2 in soil water and by faster penetration of oxygen than outflowing of CO2.

Hypo-osmotic stress-induced physiological and ion-osmoregulatory responses in European sea bass (Dicentrarchus labrax) are modulated differentially by nutritional status

Amit Kumar Sinha, AF Dasan, R Rasoloniriana, N Pipralia, R Blust, G De Boeck
Comparative Biochemistry and Physiology, Part A 181 (2015) 87–99
http://dx.doi.org/10.1016/j.cbpa.2014.11.024

We investigated the impact of nutritional status on the physiological, metabolic and ion-osmoregulatory performance of European sea bass (Dicentrarchus labrax)when acclimated to seawater (32 ppt), brackishwater (20 and 10 ppt) and hyposaline water (2.5 ppt) for 2 weeks. Following acclimation to different salinities, fish were either fed or fasted (unfed for 14 days). Plasma osmolality, [Na+], [Cl−] and muscle water contentwere severely altered in fasted fish acclimated to 10 and 2.5 ppt in comparison to normal seawater-acclimated fish, suggesting ion regulation and acid–base balance disturbances. In contrast to feed-deprived fish, fed fish were able to avoid osmotic perturbation more effectively. This was accompanied by an increase in Na+/K+-ATPase expression and activity, transitory activation of H+-ATPase (only at 2.5 ppt) and down-regulation of Na+/K+/2Cl− gene expression. Ammonia excretion rate was inhibited to a larger extent in fasted fish acclimated to low salinities while fed fish were able to excrete efficiently. Consequently, the build-up of ammonia in the plasma of fed fish was relatively lower. Energy stores, especially glycogen and lipid, dropped in the fasted fish at low salinities and progression towards the anaerobic metabolic pathway became evident by an increase in plasma lactate level. Overall, the results indicate no osmotic stress in both feeding treatments within the salinity range of 32 to 20 ppt. However, at lower salinities (10–2.5 ppt) feed deprivation tends to reduce physiological, metabolic, ion-osmo-regulatory and molecular compensatory mechanisms and thus limits the fish’s abilities to adapt to a hypo-osmotic environment.

The absence of ion-regulatory suppression in the gills of the aquatic air-breathing fish Trichogaster lalius during oxygen stress

Chun-Yen Huang, Hsueh-Hsi Lin, Cheng-Huang Lin, Hui-Chen Lin
Comparative Biochemistry and Physiology, Part A 179 (2015) 7–16
http://dx.doi.org/10.1016/j.cbpa.2014.08.017

The strategy for most teleost to survive in hypoxic or anoxic conditions is to conserve energy expenditure, which can be achieved by suppressing energy-consuming activities such as ion regulation. However, an air-breathing fish can cope with hypoxic stress using a similar adjustment or by enhancing gas exchange ability, both behaviorally and physiologically. This study examined Trichogaster lalius, an air-breathing fish without apparent gill modification, for their gill ion-regulatory abilities and glycogen utilization under a hypoxic  treatment. We recorded air-breathing frequency, branchial morphology, and the expression of ion-regulatory proteins (Na+/K+-ATPase and vacuolar-type H+-ATPase) in the 1st and 4th gills and labyrinth organ (LO), and the expression of glycogen utilization (GP, glycogen phosphorylase protein expression and glycogen content) and other protein responses (catalase, CAT; carbonic anhydrase II, CAII; heat shock protein 70, HSP70; hypoxia-inducible factor-1α, HIF-1α; proliferating cell nuclear antigen, PCNA; superoxidase dismutase, SOD) in the gills of T. lalius after 3 days in hypoxic and restricted conditions. No morphological modification of the 1st and 4th gills was observed. The air breathing behavior of the fish and CAII protein expression both increased under hypoxia. Ion-regulatory abilities were not suppressed in the hypoxic or restricted groups, but glycogen utilization was enhanced within the groups. The expression of HIF-1α, HSP70 and PCNA did not vary among the treatments. Regarding the antioxidant system, decreased CAT enzyme activity was observed among the groups. In conclusion, during hypoxic stress, T. lalius did not significantly reduce energy consumption but enhanced gas exchange ability and glycogen expenditure.

The combined effect of hypoxia and nutritional status on metabolic and ionoregulatory responses of common carp (Cyprinus carpio)

Sofie Moyson, HJ Liew, M Diricx, AK Sinha, R Blusta, G De Boeck
Comparative Biochemistry and Physiology, Part A 179 (2015) 133–143
http://dx.doi.org/10.1016/j.cbpa.2014.09.017

In the present study, the combined effects of hypoxia and nutritional status were examined in common carp (Cyprinus carpio), a relatively hypoxia tolerant cyprinid. Fish were either fed or fasted and were exposed to hypoxia (1.5–1.8mgO2 L−1) at or slightly above their critical oxygen concentration during 1, 3 or 7 days followed by a 7 day recovery period. Ventilation initially increased during hypoxia, but fasted fish had lower ventilation frequencies than fed fish. In fed fish, ventilation returned to control levels during hypoxia, while in fasted fish recovery only occurred after reoxygenation. Due to this, C. carpio managed, at least in part, to maintain aerobic metabolism during hypoxia: muscle and plasma lactate levels remained relatively stable although they tended to be higher in fed fish (despite higher ventilation rates). However, during recovery, compensatory responses differed greatly between both feeding regimes: plasma lactate in fed fish increased with a simultaneous breakdown of liver glycogen indicating increased energy use, while fasted fish seemed to economize energy and recycle decreasing plasma lactate levels into increasing liver glycogen levels. Protein was used under both feeding regimes during hypoxia and subsequent recovery: protein levels reduced mainly in liver for fed fish and in muscle for fasted fish. Overall, nutritional status had a greater impact on energy reserves than the lack of oxygen with a lower hepatosomatic index and lower glycogen stores in fasted fish. Fasted fish transiently increased Na+/K+-ATPase activity under hypoxia, but in general ionoregulatory balance proved to be only slightly disturbed, showing that sufficient energy was left for ion regulation.

The effect of temperature and body size on metabolic scope of activity in juvenile Atlantic cod Gadus morhua L.

Bjørn Tirsgaard, Jane W. Behrens, John F. Steffensen
Comparative Biochemistry and Physiology, Part A 179 (2015) 89–94
http://dx.doi.org/10.1016/j.cbpa.2014.09.033

Changes in ambient temperature affect the physiology and metabolism and thus the distribution of fish. In this study we used intermittent flow respirometry to determine the effect of temperature (2, 5, 10, 15 and 20 °C) and wet body mass (BM) (~30–460 g) on standard metabolic rate (SMR, mg O2 h−1), maximum metabolic rate (MMR, mg O2 h−1) and metabolic scope (MS, mg O2 h−1) of juvenile Atlantic cod. SMR increased with BM irrespectively of temperature, resulting in an average scaling exponent of 0.87 (0.82–0.92). Q10 values were 1.8–2.1 at temperatures between 5 and 15 °C but higher (2.6–4.3) between 2 and 5 °C and lower (1.6–1.4) between 15 and 20 °C in 200 and 450 g cod. MMR increased with temperature in the smallest cod (50 g) but in the larger cod MMR plateaued between 10, 15 and 20 °C. This resulted in a negative correlation between the optimal temperature for MS (Topt) and BM, Topt being respectively 14.5, 11.8 and 10.9 °C in a 50, 200 and 450 g cod. Irrespective of BM cold water temperatures resulted in a reduction (30–35%) of MS whereas the reduction of MS at warm temperatures was only evident for larger fish (200 and 450 g), caused by plateauing of MMR at 10 °C and above. Warm temperatures thus seem favorable for smaller (50 g) juvenile cod, but not for larger conspecifics (200 and 450 g).

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Action of Hormones on the Circulation

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

 

Introduction

This is perhaps the most difficult piece to write, unexpectedly. I have done a careful search for related material using different search phrases.  It is perhaps because of the great complexity of the topic, which is inextricably linked to sepsis, the Systemic Inflammatory Response Syndrome SIRS), and is poised differently than the neural innervation of the hormonal response and circulation, as in the previous piece.  In the SIRS mechanism, we find a very large factor in glucocorticoids, the cytokine shower (IL-1, IL-6, TNF-α), and gluconeogenesis, with circulatory changes.  In this sequence, it appears that we are focused on the arteriolar and bronchial smooth muscle architecture, the adrenal medulla, vasoconstriction and vasodilation, and another set of peptide interactions.  This may be concurrent with the other effects described.

Related articles in Pharmaceutical Intelligence Journal:

Endothelial Function and Cardiovascular Disease

Pathologist and Author: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/25/endothelial-function-and-cardiovascular-disease/

Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination?

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Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author and Curator of an Investigator Initiated Study: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator of an Investigator Initiated Study: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

Cardiovascular Disease (CVD) and the Role of Agent Alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

Curator and Investigator Initiated Study: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Innervation of Heart and Heart Rate

Writer and Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2015/02/15/innervation-of-heart-and-heart-rate/

αllbβ3 Antagonists As An Example of Translational Medicine Therapeutics

Larry H Bernstein, MD, FCAP, Reporter and curator

https://pharmaceuticalintelligence.com/2013/10/12/%CE%B1llb%CE%B23-antagonists-as-an-example-of-translational-medicine-therapeutics/

Cardiac Contractility & Myocardium Performance: Therapeutic Implications for Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC

https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differences/

Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP
and
Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-and-calcium-release-related-contractile-dysfunction-ryanopathy/

Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

Advanced Topics in Sepsis and the Cardiovascular System at its End Stage

Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-sepsis-and-the-cardiovascular-system-at-its-end-stage/

For most comprehensive Bibliography on the Ryanodine receptor calcium release channel complex and for FIGURES illustrating the phenomenon, see

Pharmacol Ther. 2009 August; 123(2): 151–177.

http://dx.doi.org:/10.1016/j.pharmthera.2009.03.006

PMCID: PMC2704947

Ryanodine receptor-mediated arrhythmias and sudden cardiac death

Lynda M. Blayney[low asterisk] and F. Anthony Lai

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2704947/

Oxidized Calcium Calmodulin Kinase and Atrial Fibrillation

Author: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/10/26/oxidized-calcium-calmodulin-kinase-and-atrial-fibrillation/

Contributions to cardiomyocyte interactions and signaling

Author and Curator: Larry H Bernstein, MD, FCAP  and Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/10/21/contributions-to-cardiomyocyte-interactions-and-signaling/

Cardiac Contractility & Myocardium Performance: Therapeutic Implications for Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Editor: Justin Pearlman, MD, PhD, FACC, Author and Curator: Larry H Bernstein, MD, FCAP, and Article Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differen/

 Action of hormones on the circulation

Limbic system mechanisms of stress regulation: Hypothalamo-pituitary-adrenocortical axis

James P. Herman, Michelle M. Ostrander, Nancy K. Muelle, Helmer Figueiredo
Prog in Neuro-Psychopharmacol & Biol Psychiatry 29 (2005) 1201 – 1213
http://dx.doi.org:/10.1016/j.pnpbp.2005.08.006

Limbic dysfunction and hypothalamo-pituitary-adrenocortical (HPA) axis dysregulation are key features of affective disorders. The following review summarizes our current understanding of the relationship between limbic structures and control of ACTH and glucocorticoid release, focusing on the hippocampus, medial prefrontal cortex and amygdala. In general, the hippocampus and anterior cingulate/prelimbic cortex inhibit stress-induced HPA activation, whereas the amygdala and perhaps the infralimbic cortex may enhance glucocorticoid secretion. Several characteristics of limbic–HPA interaction are notable: first, in all cases, the role of given limbic structures is both region- and stimulus-specific. Second, limbic sites have minimal direct projections to HPA effector neurons of the paraventricular nucleus (PVN); hippocampal, cortical and amygdalar efferents apparently relay with neurons in the bed nucleus of the stria terminalis, hypothalamus and brainstem to access corticotropin releasing hormone neurons. Third, hippocampal, cortical and amygdalar projection pathways show extensive overlap in regions such as the bed nucleus of the stria terminalis, hypothalamus and perhaps brainstem, implying that limbic information may be integrated at subcortical relay sites prior to accessing the PVN. Fourth, these limbic sites also show divergent projections, with the various structures having distinct subcortical targets. Finally, all regions express both glucocorticoid and mineralocorticoid receptors, allowing for glucocorticoid modulation of limbic signaling patterns. Overall, the influence of the limbic system on the HPA axis is likely the end result of the overall patterning of responses to given stimuli and glucocorticoids, with the magnitude of the secretory response determined with respect to the relative contributions of the various structures.

representations of the HPA axis

representations of the HPA axis

Diagrammatic representations of the HPA axis of the rat. HPA responses are initiated by neurosecretory neurons of medial parvocellular paraventricular nucleus (mpPVN), which secretes ACTH secretagogues such as corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) in the hypophysial portal circulation at the level of the median eminence. These secretagogues promote release of ACTH into the systemic circulation, whereby it promotes synthesis and release of glucocorticoids at the adrenal cortex.

When exposed to chronic stress, the HPA axis can show both response Fhabituation_ and response Ffacilitation_. FHabituation_ occurs when the same (homotypic) stressor is delivered repeatedly, and is characterized by progressive diminution of glucocorticoid responses to the stimulus. Systemic administration of a mineralocorticoid receptor antagonist is sufficient to block habituation, implying a role for MR signaling in this process. It should be noted that HPA axis habituation is highly dependent on both the intensity and predictability of the stressful stimulus. FFacilitation_ is observed when animals repeatedly exposed to one stimulus are presented with a novel (heterotypic). In chronically stressed animals, exposure to a novel stimulus results in rise in glucocorticoids that is as large as or greater than that seen in a chronic stress naıve animal. Importantly, facilitation can occur in the context of chronic stress-induced elevations in resting glucocorticoids levels, suggesting that this process involves a bypass or override of negative feedback signals.

Hippocampal regulation of the HPA axis appears to be both region- and stressor-specific. Using a sequential lesion approach, our group has noted that the inhibitory effects of the hippocampus on stress-induced corticosterone release and CRH/AVP mRNA expression are likely subserved by neurons resident in the ventral subiculum-caudotemporal CA1. In addition to spatial specificity, hippocampal regulation of the HPA axis also appears to be specific to certain stress modalities; our studies indicate that ventral subiculum lesions cause elevated glucocorticoid secretion following restraint, open field or elevated plus maze exposure, but not to ether inhalation or hypoxia.

The research posits an intricate topographical organization of prefrontal cortex output to HPA regulatory circuits. The anatomy of medial prefrontal cortex efferents may illuminate this issue. The infralimbic cortex projects extensively to the anterior bed nucleus of the stria terminalis, medial and central amygdala and the nucleus of the solitary tract, all of which are implicated in stress excitation. In contrast, the prelimbic cortex has minimal input to these structures, but projects to the ventrolateral preoptic area, dorsomedial hypothalamus and peri-PVN region, areas implicated in stress inhibition. Thus, the infralimbic and prelimbic/anterior cingulate components of the prefrontal cortex may play very different roles in HPA axis regulation. Like other limbic regions, the influence of the amygdala on the HPA axis is stressor- and region-specific. The medial amygdala shows intense c-fos induction following stressors such as restraint, swimming, predator exposure and social interaction.

Despite the prominent involvement of the hippocampus, medial prefrontal cortex and amygdala in HPA axis regulation, there is limited evidence of direct innervation of the PVN by these structures. Rather, these regions appear to project to a number of basal forebrain, hypothalamic and brainstem cell populations that in turn innervate the medial parvocellular PVN. Thus, in order to access principle stress effector neurons, information from the limbic system requires an intermediary synapse. In the bed nucleus of the stria terminalis and hypothalamus, the majority of these intermediary neurons are GABAergic. For example, the bed nucleus of the stria terminalis, ventrolateral preoptic area, dorsomedial hypothalamic nucleus and peri-PVN region all contain rich populations of neurons expressing the GABA marker glutamatic acid decarboxylase (GAD) 65/67.

The organization of the peri-PVN cell groups is particularly interesting. In the case of the ventral subiculum and to a lesser extent, the medial prefrontal cortex, terminal fields can be observed in the immediate surround of the PVN, corresponding to areas containing substantial numbers of GABA neurons. Importantly, dendrites of PVN neurons are largely confined within the nucleus proper, indicating that limbic afferents are unlikely to interact directly with the PVN neurons themselves. The peri-PVN GABA neurons are activated by glutamate, and likely express glutamate receptor subunits. These neurons also up-regulate GAD65 mRNA following chronic stress, commensurate with involvement in long-term HPA regulation. Injections of a general ionotroptic glutamate receptor antagonist into the PVN surround potentiates glucocorticoid responses to restraint, consistent with blockade of glutamate excitation of these GABA neurons. The data are consistent with an interaction between the excitatory limbic structures and inhibitory PVN-regulatory cells at the level of the PVN surround.

Brainstem stress-modulatory pathways likely relay excitatory information to the PVN. For example, the nucleus of the solitary tract provides both catecholaminergic (norepinephrine) and non-catecholaminergic (e.g., glucagon-like peptide-1 (GLP-1) input to the medial parvocellular. Norepinephrine is released into the PVN following stress and is believed to activate CRH neurons via alpha-1 adrenergic receptors. The role of this pathway is thought to be associated with systemic stressors, as selective destruction of PVN norepinephrine input using anti-dopamine beta hydroxylase-saporin conjugate blocks responses to 2-deoxy-glucose but not restraint.  In contrast, blockade of central GLP-1 receptors using exendin 9–36 markedly inhibits responsiveness to both lithium chloride and novelty, suggesting that this non-catecholaminergic cell population may play a more general role in stress integration.

The existence of these putative two-neuron circuits lends important insight into the nature of stress information processing. Anatomical data support the hypothesis that the vast majority of medial prefrontal cortex and ventral subicular inputs to subcortical stress relays are glutamate-containing. As can be appreciated, pyramidal cells of the medial prefrontal cortex and subiculum richly express mRNA encoding vesicular glutamate transporter-1 (VGlut1), a specific marker of glutamate neurons. Combined retrograde tracing/in situ hybridization studies performed in our lab indicate that the vast majority of cortical and hippocampal afferents to PVN-projecting regions (e.g., bed nucleus of the stria terminalis, dorsomedial hypothalamus, ventrolateral medial preoptic area) indeed contain VGlut1, verifying a glutamatergic input to these areas. In contrast, the majority of amygdalar areas implicated in stress regulation express glutamic acid decarboxylase (GAD) 65 or 67 mRNA, suggesting a GABAergic phenotype; indeed, the vast majority of medial and central amygdaloid projections to PVN relays are GABAergic.

representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus

representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus

Diagrammatic representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus. The medial prefrontal cortex (mPFC) subsumes neurons of the prelimbic (pl), anterior cingulate (ac) and infralimbic cortices (il), which appear to have different actions on the HPA axis stress response. The pl/ac send excitatory projections (designated as dark circles, filled line with arrows) to regions such as the peri-PVN zone and bed nucleus of the stria terminalis (BST), both of which send direct GABAergic projections to the medial parvocellular PVN (delineated as open circles, dotted lines ending in squares). This two-neuron chain is likely to be inhibitory in nature. In contrast, the infralimbic cortex projects to regions such as the nucleus of the solitary tract (NTS), which sends excitatory projections to the PVN, implying a means of PVN excitation from this cortical region. The ventral subiculum (vSUB) sends excitatory projections to numerous subcortical regions, including the posterior BST, peri-PVN region, ventrolateral region of the medial preoptic area (vlPOA) and ventrolateral region of the dorsomedial hypothalamic nucleus (vlDMH), all of which send GABAergic projections to the PVN and are likely to communicate transsynaptic inhibition. The medial amygdaloid nucleus (MeA) sends inhibitory projections to GABAergic PVN-projecting populations, such as the BST, vlPOA and peri-PVN, eliciting a transsynaptic disinhibition. A similar arrangement likely exists for the central amygdaloid nucleus (CeA), which sends GABAergic outflow to the ventrolateral BST and to a lesser extent, the vlDMH. The CeA also projects to GABAergic neurons in the NTS, which may disinhibit ascending projections to the PVN.

Inotropes and vasopressors: more than haemodynamics!

Hendrik Bracht, E Calzia, M Georgieff,  J Singer, P Radermacher and JA Russell
British Journal of Pharmacology (2012) 165 2009–2011
http://dx.doi.org:/10.1111/j.1476-5381.2011.01776.x

Circulatory shock is characterized by arterial hypotension requiring fluid resuscitation combined with inotropes and/or vasopressors to correct the otherwise life-threatening impairment of oxygen supply to peripheral tissues. Catecholamines represent the current therapeutic choice, but this standard is only based on empirical clinical experience. Although there is evidence that some catecholamines may be better than others, it is a matter of debate which one may be the most effective and/or the safest for the different situations. In their review in this issue of the British Journal of Pharmacology, Bangash et al. provide an overview of the pharmacology as well as the available clinical data on the therapeutic use of endogenous catecholamines, their synthetic derivatives and a range of other agents (vasopressin and its analogues, PDE inhibitors and levosimendan). The authors point out that, despite well-established receptor pharmacology, the clinical effects of these treatments are poorly understood. Hence, further investigations are essential to determine which catecholamine, or, in a broader sense, which alternative vasopressor and/or inotrope is the most appropriate for a particular clinical condition.

LINKED ARTICLES   This article is a commentary on Bangash et al., pp. 2015–2033 of this issue and is commented on by De Backer and Scolletta, pp. 2012–2014 of this issue. To view Bangash et al. visit http://dx.doi.org/10.1111/j.1476-5381.2011.01588.x   and to view De Backer and Scolletta visit http://dx.doi.org/10.1111/j.1476-5381.2011.01746.x

In the present issue of the British Journal of Pharmacology, Bangash et al. (2012) review the pharmacology as well as the available clinical data on the therapeutic use of various inotropes and vasopressor agents used for the hemodynamic management of (septic) shock. By definition, circulatory shock is characterized by arterial hypotension that necessitates immediate intervention to maintain the balance of tissue oxygen supply and demand. In practice, the longer and the more frequent periods of hypotension are present in a patient, the less likely is survival, and early aggressive resuscitation is associated with improved outcome. Besides fluid administration to increase the circulating blood volume, in most cases, vasoactive drugs are required to restore an adequate perfusion pressure, and up to now, catecholamines represent the current therapeutic choice. According to their pharmacological profile, catecholamines are traditionally used for their predominant inotropic, vasodilating or constrictor effects.

Clinicians should not forget two fundamental aspects of catecholamine action. First, because of the ubiquitous presence of adrenoceptors, endogenous catecholamines. as well as their synthetic derivatives, have pronounced effects on virtually all tissues (many of which were described several years ago), in particular on the immune system (van der Poll et al., 1996; Flierl et al., 2008), on energy metabolism (Cori and Cori, 1928; Bearn et al., 1951) and on gastrointestinal motility (McDougal and West, 1954). Second, the adrenoceptor density and responsiveness to catecholamines are markedly altered by both the underlying disease and the ongoing catecholamine. Bangash et al. (2012) have to be commended that they not only describe the various endogenous catecholamines and their synthetic derivatives but also thoroughly discuss possible alternatives, such as vasopressin and its analogues, PDE inhibitors and levosimendan.

Inhibitory effects of cortisone and hydrocortisone on human Kv1.5 channel currents

Jing Yu, Mi-Hyeong Park, Su-Hyun Jo
Eur J Pharmacol 746 (2015) 158–166  http://dx.doi.org/10.1016/j.ejphar.2014.11.007

Glucocorticoids are the primary hormones that respond to stress and protect organisms from dangerous situations. The glucocorticoids hydrocortisone and its dormant form, cortisone, affect the cardiovascular system with changes such as increased blood pressure and cardioprotection. Kv1.5 channels play a critical role in the maintenance of cellular membrane potential and are widely expressed in pancreatic β-cells, neurons, myocytes, and smooth muscle cells of the pulmonary vasculature. We examined the electrophysiological effects of both cortisone and hydrocortisone on human Kv1.5 channels expressed in Xenopus oocytes using a two-microelectrode voltage clamp technique. Both cortisone and hydrocortisone rapidly and irreversibly suppressed the amplitude of Kv1.5 channel current with IC50 values of 50.2 + 74.2 μM and 33.4 + 73.2 μM, respectively, while sustained the current trace shape of Kv1.5 current. The inhibitory effect of cortisone on Kv1.5 decreased progressively from – 10mV to +30 mV, while hydrocortisone’s inhibition of the channel did not change across the same voltage range. Both cortisone and hydrocortisone blocked Kv1.5 channel currents in a non-use-dependent manner and neither altered the channel’s steady-state activation or inactivation curves. These results show that cortisone and hydrocortisone inhibited Kv1.5 channel currents differently. Kv1.5 channels were more sensitive to hydrocortisone than to cortisone.

In conclusion, cortisone and hydrocortisones rapidly and irreversibly blocked human Kv1.5 channels expressed in Xenopus oocytes in a closed state without altering activation and inactivation gating. These data provide a possible mechanism for GC effects on the cardiovascular system. The detailed mechanism of the interaction between GCs and human Kv1.5 channels merits further exploration.

Inflammasome and cytokine blocking strategies in autoinflammatory disorders

Monika Moll, Jasmin B. Kuemmerle-Deschner
Clinical Immunology (2013) 147, 242–275 http://dx.doi.org/10.1016/j.clim.2013.04.008

Autoinflammatory disorders are characterized by usually unprovoked recurrent episodes of features of inflammation caused by activation of the innate immune system. Many autoinflammatory disorders – the monogenetic defects in particular – are associated with alterations of inflammasomes. Inflammasomes are complex multimolecular structures, which respond to “danger” signals by activation of cytokines. Among these, IL-1 is the key player of the innate immune response and inflammation. Consequently, IL-1 blocking strategies are specific pathway targeting therapies in autoinflammatory diseases and applied in CAPS, colchicine-resistant FMF, TRAPS, HIDS and DIRA. A number of rare genetic disorders involve inflammasome malfunction resulting in enhanced inflammatory response. IL-1 inhibition to date is the most successful specific therapy in autoinflammatory disorders. Here, current treatment strategies in autoinflammatory disorders are reviewed with a focus on inflammasome and cytokine inhibition.

Autoinflammatory disorders have been defined as “clinical disorders marked by abnormally increased inflammation, mediated predominantly by the cells and molecules of the innate immune system.”  This means that in autoinflammatory disorders autoantibodies or antigen related T-cells are usually absent. These are features of the adaptive immune system and found in autoimmune diseases.
In general, autoinflammatory disorders are characterized by a large spectrum of rather non-specific systemic and organ-specific signs and symptoms of inflammation. In some diseases specific symptoms are observed like hearing loss in Muckle–Wells syndrome or CNS-disease in NOMID/CINCA. Most autoinflammatory disorders are associated with high levels of serum amyloid A (SAA) during inflammatory attacks and high risk of life-threatening amyloidosis. In most cases the disease will start in infancy and childhood. Only rarely primary manifestations in adulthood are reported.
Because recurrent fevers have been the most prominent feature of this group of diseases, historically they have been summarized under the term “hereditary periodic fever syndromes”.  With the deeper understanding of the underlying pathophysiologic mechanisms on the genetic and cellular level, the more comprehensive term “autoinflammatory syndromes”.
Along with the detection of the genetic origin of the autoinflammatory disorders, the cellular pathomechanism leading to the resulting inflammation has been described. A number of genes, which are affected by mutations in autoinflammatory disorders, encode proteins forming intracellular complexes called inflammasomes. External and endogenous “dangers” are recognized by these “danger sensors” and are able to induce an inflammatory reaction. Microbial components from infectious agents such as LPS, flagellin, lipoteichoic acid from bacteria, peptidoglycan or double-stranded DNA from viruses, or inorganic crystalline structures such as uric acid crystals, display pathogen-associated molecular patterns (PAMPs). These and endogenous damage-associated molecular patterns (DAMPs) like heat-shock proteins, the chromatin-associated protein high-mobility group box 1 (HMGB1), hyaluronan fragments, ATP, uric acid, and DNA which are released with cellular waste and injury stimulate the inflammasome. Also, the myeloid related proteins MRP8 and 14 (also known as S100A8 and S100A9) which are used as biomarkers, belong to the group of DAMPs. In addition to PAMPs and DAMPs, the inflammasome may interact with and be stimulated by proteins such as pyrin, proline–serine–threonine phosphatase interacting protein 1 (PSTPIP1), mevalonate kinase (MK) and NLRP7. All of these may also be altered in structure and function by monogenetic mutations.
As a consequence of inflammasome activation, a large variety of cytokines are produced and released by cells of the innate immune system (monocytes, macrophages, dendritic cells). They include the IL-1 family (IL-1, IL-18, IL-33), the TNF family (TNF-α, LT-α), the IL-6 family (IL-6, IL-11), the IL-17 family (IL-17A, IL-25), and type 1 IFNs (IFN-α, IFN-β). These cytokines play redundant roles depending on the cause and pathway of inflammation in the respective disease. Therefore, therapeutic strategies targeting only one cytokine should be expected to be inadequate to treat inflammatory disorders. However, improvement observed in diabetes mellitus Type 2 after blockade of IL-1 indicates that targeting one cytokine, even in a polygenic, complex inflammatory disorder, may cause beneficial effects. Regarding the inflammatory pathogenesis involved in the disease, Goldbach–Mansky and co-workers have classified the monogenetic autoinflammatory disorders as IL-1 mediated (CAPS and DIRA), partially IL-1 mediated (FMF, HIDS, PAPA) and mediated by other pathways (TRAPS, Blau-syndrome, Majeed’s syndrome, cherubism and IL-10 receptor deficiency).

Intracellular signaling pathways and therapeutic targets in autoinflammatory diseases. In autoinflammatory diseases, complex intracellular pathways lead to activation of the inflammatory response, particularly IL-1β activation and release, but also induction of NFκB and TNFα. Several mechanisms may activate the inflammasome, one crucial step in the IL-1 pathway. These include DAMPs (1), K+-efflux (2), activation of ROS (3) by ATP, anorganic crystals, membrane perturbation and proteases which are released from lysosomes damaged by β-amyloid, and heat shock proteins (4). NFκB may be induced by PAMPs via toll like receptors (5), IL-1β-signaling (6) or UPR (7). Activated NFκB eventually leads to the release of pro-inflammatory cytokines like IL-1, IL-6 and TNFα (8). Most of these steps to activation have been identified as targets for anti-inflammatory therapies, which are either already used in clinical practice or still experimental. IL-1- (a), TNF- (b), and IL-6 (c) inhibition are established safe and effective treatment strategies in many autoinflammatory diseases. Thalidomide (d) probably inhibits activation of IκB and is also part of routine treatment. Still experimental strategies include inhibition of PAMPs (e), DAMPs (f), potassium efflux (g), ROS by antioxidants (h), heat shock proteins (i), or caspase-1 (k). Caspase-inhibitors have entered clinical trials.

Colchicine has been used for the treatment of inflammatory disorders for centuries. Colchicine is effective in gout, but also in Behcet’s disease and FMF, where it is able to prevent amyloidosis. The drug affects many cell types and accumulates preferentially in neutrophils. Although its mode of action is still unclear it has microtubule destabilizing properties which may be part of its effects. Additional effects such as alteration of adhesion molecule expression, chemotaxis, and ROS generation also impact inflammation. Colchicine is generally tolerated well. However gastrointestinal, hematologic, and neuromuscular side-effects occur, when the administered dose is too high.

Inflammasome activation by heat shock proteins may be prevented by direct inhibition of HSP. HSP90 inhibition was effective in reducing gout-like arthritis in an animal model. Targeting caspase-1 (caspase-1-inhibitors) may be a strategy which has even greater potential in the treatment of autoimmune diseases and autoinflammatory disorders. IL-1 converting enzyme/caspase inhibitor VX-765 was able to inhibit IL-β-secretion in LPS-stimulated cells from FCAS and control subjects. A new IL-1 inhibitor, gevokizumab or Xoma 052 has entered clinical pilot trials. Therapeutic targets particularly for the protein-misfolding autoinflammatory diseases could be chemical chaperones and drugs that stimulate autophagy. Also inhibiting the signaling molecules that mediate the UPR activation which causes activation of the innate immune system and exacerbate inflammation could be a target.

To date IL-1 blockade is the most effective therapy in most monogenetic autoinflammatory diseases — in intrinsic and in extrinsic inflammasom-opathies. The most favorable effects are seen in the treatment of cryopyrin associated periodic syndromes like FACS, MWS and CINCA. But IL-1-blockade is also effective in other diseases like DIRA, TRAPS, PFAPA, colchicine-resistant FMF etc. IL-1 inhibition also has a role in multifactorial and common autoinflammatory diseases like diabetes, gout and artherosclerosis.

Endothelin—Biology and disease

Al-karim Khimji, Don C. Rockey
Cellular Signalling 22 (2010) 1615–1625
http://dx.doi.org:/10.1016/j.cellsig.2010.05.002

Endothelins are important mediators of physiological and pathophysiologic processes including cardiovascular disorders, pulmonary disease, renal diseases and many others. Additionally, endothelins are involved in many other important processes such as development, cancer biology, wound healing, and even neurotransmission. Here, we review the cell and molecular biology as well as the prominent pathophysiological aspects of the endothelin system.

Endothelin-1 (ET-1) was originally isolated from porcine aortic endothelial cells  and is a 21 amino acid cyclic peptide, with two disulphide bridges joining the cysteine amino acids (positions 1–15 and 3–11) at the N-terminal end and hydrophobic amino acids at the c-terminal end of the peptide (Fig. 1). The C-terminal end contains the amino acids that bind to the receptor, the N-terminal end determines the peptide’s binding affinity to the receptor (see Fig. 1). There appear to be at least 2 other endothelin isoforms including endothelin-2 (ET-2) and endothelin-3 (ET-3), which differ from ET-1 in two and six amino acid residues, respectively.

Endothelin (ET) structure

Endothelin (ET) structure

Endothelin (ET) structure. Endothelin is a 21 amino acid cyclic peptide, with two disulphide bridges joining the cysteine residues at positions 1–15 and 3–11. The C-terminal end containsamino acids that appear tomediate receptor binding,while the N-terminal residues determine the peptide’s binding affinity to the receptor. The amino acids highlighted in black in panels (b) and (c) show differences in ET-2 and ET-3 compared to ET-1. As can be seen, the remainder of the primary sequence of the different family members is identical.

Endothelin-1 biosynthetic pathway

Endothelin-1 biosynthetic pathway

Endothelin-1 biosynthetic pathway. Preproendothelin mRNA is synthesized via transcriptional activation of the preproendothelin gene. The translational product is a 203-amino acid peptide known as preproendothelin, which is cleaved at dibasic sites by furin-like endopeptidases to form big endothelins. These biologically inactive, 37- to 41-amino acid intermediates, are cleaved at Trp21–Val 22 by a family of endothelin-converting enzymes (ECE) to produce mature ET-1. The pathway for endothelin-2 and -3 is presumed to be similar.

The endothelin peptides are produced through a set of complex molecular processes. Preproendothelins are synthesized via transcriptional activation of the preproendothelin gene, which is regulated by c-fos and c-jun, nuclear factor-1, AP-1 and GATA-2. The translational product is a 203-amino acid peptide known as preproendothelin which is cleaved at dibasic sites by furin-like endopeptidases to form big endothelins. These biologically inactive 37- to 41-amino acid intermediates are cleaved at Trp21–Val 22 by a family of endothelin-converting enzymes (ECE) to produce mature ET-1.

Three isoforms of ECE have been reported, namely ECE-1, ECE-2 and ECE-3; ECE-1 and ECE-2 are most prominent. (Endothelin receptors are widely distributed in many different tissues and cells, there is a marked difference in cell and tissue distribution patterns between the two receptor subtypes i.e. ETA and ETB. [ET Receptors: Endothelial cells -ETB Vascular tone, clearance of circulating ET-1]).  ECEs belong to the M13 group of proteins—which is a family that includes neutral endopeptidases, kell blood group antigens (Kell), a peptide from phosphate regulating gene (PEX), X-converting enzyme (XCE), “secreted” endopeptidases, and the ECEs. M13 family members contain type II integral membrane proteins with zinc metalloprotease activity, and their function is inhibited by phosphoramidon. Four variants of ECE-1 have been reported in humans, namely ECE-1a, ECE-1b, ECE-1c and ECE-1d which are a result of alternate splicing of ECE-1mRNA. ECE-1 appears to be localized in the plasma cell membrane and its optimal activity is atpH7; it processes big ETs both intracellularly and on the cell surface. It is distributed predominantly in smooth muscle cells. ECE-1 can also hydrolyze other proteins including bradykinin, substance P, and insulin. ECE-2 is localized to the trans-Golgi network and is expressed abundantly in neural tissues and endothelial cells. Its optimal activity is at pH5; the acidic activity marks ECE-2 as an intracellular enzyme. Substrate selectivity experiments indicate that both ECE-1 and ECE-2 show preference for big ET-1 over big ET-2 or big ET-3.

Although there has been controversy about the precise repertoire of endothelin receptors, it appears that the endothelins exert their actions through two major receptor subtypes known as ETA and ETB receptors. ETA and ETB receptors belong to the superfamily of G-protein coupled receptors and contain seven transmembrane domains of 22–26 hydrophobic amino acids among approximately 400 total amino acids. The ETA receptor is found predominantly in smooth muscle cells and cardiac muscles, whereas the ETB receptor is abundantly expressed in endothelial cells.

ET-1 signaling is extremely complicated and ET receptor activation leads to diverse cellular responses through interaction in a chain of pathways that includes the G-protein-activated cell surface receptor, coupling G-proteins and phospholipase (PLC) pathway and other G protein-activated effectors. In one of the canonical signaling pathways, ETA induced activation of phospholipase C leads to the formation of inositol triphosphate and diacylglcerol from phosphatidylinositol. Inositol 1,4,5 triphosphate (IP3) then diffuses to specific receptors on the endoplasmic reticulum and releases stored Ca2+ into the cytosol. This causes a rapid elevation in intracellular Ca2+, which in turn causes cellular contraction and then vasoconstriction; the vasoconstrictive effects of ET persist despite dissociation of ET-1 from the receptor, perhaps because the levels of intracellular calcium remain elevated or because endothelin signaling pathways remain activated for prolonged time periods.

Endothelin signaling – smooth muscle cells

Endothelin signaling – smooth muscle cells

Endothelin signaling – smooth muscle cells. ET receptor stimulation leads to diverse cellular responses in a chain of pathways that include the G protein bg activation. This is followed by activation of a variety of different downstream cascades. For example, shown on the left, ETA induced activation of phosphatidyl inositol specific phospholipase C (PI-PLC) leads to the formation of inositol triphosphate (IP3) and diacylglcerol (DAG) from phosphoinositol 4,5 bisphosphate (PIP2). Inositol 1, 4, 5 triphosphate (IP3) then diffuses to specific receptors on the endoplasmic reticulum and releases stored Ca2+ into the cytosol. This causes a rapid elevation in intracellular Ca2+, which in turn causes cellular contraction

Endothelin signaling – endothelial cells.

Endothelin signaling – endothelial cells.

Endothelin signaling – endothelial cells. ET-1 stimulates NO production in endothelial cells by activation of endothelial cell NO synthase (eNOS). This occurs via ET-1’s activation of the ET-B receptor and the PI3-K/Akt pathway, which in turn stimulates phosphorylation of eNOS, with subequent conversion of L-arginine to L-citrulline and at the same time, generating NO. In addition shear stress, G-protein coupled receptors (GPCR), transient receptor potential channel (TRPC) and receptor tyrosine kinase (RTK) are also activators of eNOS. As a result, NO diffuses to stellate cell, where it directly activates the heme moiety of soluble guanylate cyclase, leading to the production of cyclic GMP. Intracellular cyclic GMP leads to activation of protein kinase G (PKG) resulting in relaxation of stellate cells – offsetting ET’s contractile effect on stellate cells.

The plasma levels of endothelin do not correlate with either the presence of essential hypertension or its severity, presumably, due to the fact that endothelin appears to be biologically active in a paracrine or autocrine fashion (i.e., rather than in an endocrine fashion. Systemic administration of ET-1 in low doses produces a modest increase in blood pressure which is normalized by selective ETA receptor blockade. In experimental models, long-term infusion with ET-1 leads to stroke and renal injury, which can be prevented with long-term administration of selective ETA receptor antagonists. Apart from its direct vasoconstrictor effects, mediated by smooth muscle cell contraction in the arterial system, ET-1 also indirectly enhances the vasoconstrictor effects of other neurohumoral and endocrine factors and may potentiate essential hypertension via this mechanism. For example, ET-1 induces conversion of angiotensin I to angiotensin II in in vitro models and stimulates adrenal synthesis of epinephrine and aldosterone. Thus there is cross-talk between the endothelin and renin–angiotensin–aldosterone systems—to synergistically act to facilitate vasoconstriction. In aggregate, the data suggest that dysregulation of the endothelin system contributes to multisystem complications of hypertension such as progressive renal disease, cerebrovascular diseases, atherosclerosis, and cardiac disease.

ET-1 in the renal system is synthesized in vascular endothelial cells and epithelial cells of the collecting ducts. Both ET receptors are present in renal vasculature and epithelial cells where ETB is the predominant receptor type. Renal vasculature is relatively more sensitive to the vasoconstrictive effects of ET-1 than any other vasculature and it causes constriction of both afferent and efferent renal arterioles.

ET-1 administration in humans significantly reduces renal blood flow, glomerular filtration rate and urine volume. In addition to its hemodynamic effects, ET-1 system is also involved in salt and water reabsorption, acid-base balance, promotion of mesangial cell growth and activation of inflammatory cells. ET-1 has been implicated in the pathophysiology of acute renal injury, chronic renal failure as well as renal remodeling. Transgenic mice overexpressing ET-1 develop glomerulosclerosis, interstitial fibrosis and reduced renal function. Increased ET-1 and ET receptor upregulation has been described in various animal models of acute renal injury and also in patients with chronic renal failure. Additionally, plasma ET-1 levels have been shown to correlate with the severity of chronic renal failure.

ET-1 is produced and released by airway epithelial cells, macrophages, and pulmonary vascular endothelial cells. Endothelin receptors are similarly widely distributed in airway smooth muscle cells, the pulmonary vasculature, and in the autonomic neuronal network lining tracheal muscles. ET-1 has a potent bronchoconstrictor effect.  In animal models, intravenous ET-1 injection led to a dose-dependent increase in airway resistance. The increase in airway resistance is in part due to enhanced production of thromboxanes with subsequent activation of thromboxane receptors and smooth muscle cell proliferation. The ET system has been emphasized in a number of pulmonary disorders, including asthma, cryptogenic fibrosing alveolitis, and pulmonary hypertension. Increased lung vasculature ET-1 immunoreactivity has been reported in both animals and patients with pulmonary hypertension and increases in ET-1 immunoreactivity correlate with the degree of pulmonary vascular resistance, disorders such as pulmonary hypertension, myocardial infarction, heart failure, neoplasia, vascular disorders, wound healing, and many others.

Endothelin and endothelin antagonism: Roles in cardiovascular health and disease

Praveen Tamirisa, William H. Frishman, and Anil Kumar
Am Heart J 1995;130:601-10

Endothelin is a naturally occurring polypeptide substance with potent vasoconstrictive actions. It was originally described as endotensin or endothelial contracting factor in 1985 by Hickey et al., who reported on the finding of a potent stable vasoconstricting substance produced by cultured endothelial cells. Subsequently, Yanagisawa et al. isolated and purified the substance from the supernatant of cultured porcine aortic and endothelial
cells and then went on to prepare its complementary deoxyribonucleic acid (cDNA). This substance was renamed endothelin.

Endothelin is the most potent vasoconstrictor known to date. Its chemical structure is closely related to certain neurotoxins (sarafotoxins) produced by scorpions and the burrowing asp (Atractaspis engaddensis).  Endothelins have now been isolated in various cell lines from several organisms. They are now considered to be autocoids or cytokines 4 because of their wide distribution, their expression during ontogeny and adult life, their primary role as intracellular factors, and the complexity of their biologic effects.

The superfamily of endothelins and sarafotoxins have two main branches with four members each. Endothelin is a polypeptide consisting of 21 amino acids. There are three closely related isoforms endothelin-1, endothelin-2, and endothelin-3 (ET1, ET2, and ET3, respectively), which differ in a few of the amino acid constituents. The fourth member, called ET4 or vasoactive intestinal constrictor, is considered to be the murine form ofET2. The endothelin molecules have several conserved amino acids, including the last six carboxyl (C)-terminal amino acids and four cysteine residues, which form two intrachain disulfide bonds between residues 1 and 15 and 3 and 11. These residues may have biologic implications particularly in relation to three dimensional structure and function. The main differences in the endothelin isopeptides reside in their amino (N)-terminal segments. There is a very high degree of sequence similarity between the two branches (approximately 60%) and within the constituent members of a branch (71% to 95%).

Endothelin has been demonstrated to be produced from endothelial and nonendothelial cells. The synthesis of endothelins parallels that of the various peptide hormones in that a precursor polypeptide is sequentially cleaved to generate the active form. Recently, endothelin-converting enzyme (ECE) was cloned. ECE acts at an essential step in the production of active forms of endothelins. The fully formed molecule is then broken down into inactive peptides by as yet uncharacterized proteases. Some candidates are the lysosomal protective protein (deamidase) and enkephalinase (neutral endopeptidase EC 24.11). The regulation of endothelin production occurs predominantly at the levels of transcription and translation. No storage
vesicles containing endothelin have been identified. The genes for the various endothelin isoforms have been sequenced and are found to be scattered in different chromosomes. Current evidence suggests that they arose from a common ancestor by exon duplication.

Factors known to release endothelinThrombinTransforming growth factor-~Arginine vasopressinHypoxia

Phorbol ester

Glucose

Angiotensin II

Cyclosporin

Insulinlike growth factor

Bombesin

Cortisol

Low-density lipeprotein cholesterol

Hypercholesterolemia

Changes in shear stress on vascular wall

Receptor affinities
Receptor Affinity
ETA ET1 > ET2 > ET3
ETB ET1 = ET2 = ET3
ETC ET3 > ET1
Intracellular signal transduction pathways activated by endothelins (ETs)

Intracellular signal transduction pathways activated by endothelins (ETs)

Intracellular signal transduction pathways activated by endothelins (ETs). Activated ET receptor stimulates phospholipase C (PLC) and phospholipase A2 (PLA2). Activated ET receptor also stimulates voltage-dependent calcium channels (VDC) and probably receptor-operated calcium channel (ROC). Inositol triphosphate (IP3) elicits release of calcium ion from caffeine-sensitive calcium store. Protein kinase C (PKC) activated by diacylglycerol (DG) sensitizes contractile apparatus. Increased concentration of intracellular free calcium ion ([Ca2+]i induces contraction. Cyclooxygenase products (prostacyclin [PGI2], prostaglandin E2 [PGE2], and thromboxane A2 [TXA2]) modify contraction. G, G protein; IP2, inositol biphosphate; IP3, inositol triphosphate; PIP2, phosphatidyl inositol biphosphate. (From Masaki T et al. Circulation 1991;84: 1460.)

Systemic hypertension. Endothelin is the most potent vasoconstrictor known to date and has an exceptionally long duration of physiologic action. The influence of endothelin in maintaining normal blood pressure and its role in the cause of systemic hypertension remain unclear. Intravenous injections of endothelin in animals cause a transient decrease in systolic blood pressure (ETB) followed by a prolonged pressor response (ETA). The vasoconstrictor action is mediated by ETA receptors in the vascular smooth muscle, whereas the predominant vasodilation effect is mediated by the ETB receptors on the endothelial cells that cause release of prostacyclin and nitric oxide. Therefore the overall predominant hemodynamic effect of endothelin in a given organ depends on the receptor type being stimulated, its location, and its relative abundance.

Angiotensin II has been found to increase endothelin concentrations in vitro from endo thelial cells, suggesting one mechanism by which angiotensin-converting-enzyme (ACE) inhibition could function in vivo. ACE inhibitors also can indirectly interfere with endothelin: increased concentrations of bradykinin decrease endothelin release (by acting through bradykinin 2 receptors, stimulation of which cause increased nitric oxide release). ACE inhibitors can cause regression of intimal hyperplasia, whereas other antihypertensive drugs are ineffective in this regard.

Myocardial ischemia. Myocardial ischemia can enhance the release of endothelin by cardiomyocytes and increase its vasoactive effects. Infusion of the ET1 isoform directly into the coronary circulation of animals results in the development of myocardial infarction, with impaired ventricular functioning and the development of arrhythmias. Endothelin has been shown to lower the threshold for ventricular fibrillation in dogs. An increase in ET1 has been observed in cardiac tissue after experimental myocardial infarction in rats, and pretreatment with an antiendothelin ϒ-globulin in this model can reduce infarct size by as much as 40%. Infusion of ETA receptor antagonist drugs before an ischemic insult can also reduce infarct size in animals.

Plasma endothelin concentrations can predict hemodynamic complications in patients with myocardial infarction. Patients with the highest plasma endothelin concentrations after myocardial infarction have the highest creatine phosphokinase (CPK) and CPK MB-isoenzyme concentrations and the lowest angiographically determined ejection fractions.

Left ventricular function and congestive heart failure. Endothelin exhibits potent inotropic activity in isolated hearts, cardiac muscle strips, isolated cells, and instrumented intact animals. High-affinity receptors for endothelin have been demonstrated in the atria and the ventricles. Intravenous administration of the ET1 isoform produces delayed prolonged augmentation of left ventricular performance in addition to its biphasic vasoactive effects of transient vasodilation followed by sustained vasocontraction.

Endothelin is a potent secretogogue of atrial natriuretic factor, which is a naturally occurring antagonist of endothelin. The ETA receptor appears to mediate endothelin’s actions of vasoconstriction and the stimulation of atrial natriuretic factor secretion, and the ETB receptor mediates endothelin-induced vasodilation and activation of the renin-angiotensin-aldosterone system. Urinary water excretion is mediated through both receptors, but sodium excretion is mediated through the ETA receptor.

Increased concentrations of endothelin described in patients with congestive heart failure are predictive of increased mortality risk. It also has been suggested that increased concentrations of endothelin may play an important role in the increased systemic vascular resistance observed in congestive heart failure.

There is early clinical evidence that treatment with ETA receptor antagonists and ECE inhibitors can influence favorably the course of human heart failure.  ACE inhibitors may also benefit patients with heart failure because of their antiendothelin actions.

Pulmonary hypertension. Expression of ET1 in the lung has been studied by immunocytochemistry and hybridization in situ in specimens from patients with pulmonary hypertension of primary or secondary causes. In contrast to normal lung, specimens from patients with pulmonary hypertension exhibit abundant ET2 immunostaining, particularly over endothelium of markedly hypertrophied muscular pulmonary arteries and plexogenic lesions. Endothelin has been suggested as a potent vasoconstrictor and growth-promoting factor in the pathophysiologic pathophysiologic mechanisms of pulmonary hypertension.

Ventricular and vascular hypertrophy. Endothelin increases DNA synthesis in vascular smooth-muscle ceils, cardiomyocytes, fibroblasts, glial cells, mesangial cells, and other cells; causes expression of protooncogenes; causes cell proliferation; and causes hypertrophy. It acts in synergy with various factors such as transforming growth factor, epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor and insulin to potentiate cellular transformation and replication. This synergy suggests that all of these factors act through common pathways involving PKC and cyclic adenosine monophosphate. Endothelin per se may not be a direct mediator of angiogenesis but may function as a comitogenic factor.

Neointima formation after vascular wall trauma. The efficacy of coronary angioplasty is limited by the high incidence of restenosis. ET1 induces cultured vascular smooth-muscle cell proliferation by activation of the ETA-receptor subtype, a response that normally is attenuated by an intact, functional endothelium. In addition, ET1 also induces the expression and release of several protooncogenes and growth factors that modulate smooth-muscle cell migration, proliferation, and matrix formulation. In addition to inhibiting smooth-muscle cell proliferation in vitro, endothelin-receptor antagonism with SB 209670 ameliorates the degree of neointima formation observed after rat carotid artery angioplasty. The observations raise the possibility that ET1 antagonists will serve as novel therapeutic agents in the control of restenosis.

Nonspecific endothelin antagonists
ECE inhibitorsAngiotensin-converting-enzyme inhibitorsAngiotensin II receptor blocking agentsCalcium-entry blocking agentsPotassium-channel opening agentsAdenosineNitroglycerin

 

 

 

 

SUMMARY

Endothelin is the most potent mammalian vasoconstrictor yet discovered. Its three isoforms play leading roles in regulating vascular tone and causing mitogenesis. The isoforms bind to two major receptor subtypes (ETA and ETB), which mediate a wide variety of physiologic actions in several organ systems. Endothelin may also be a disease marker or an etiologic factor in ischemic heart disease, atherosclerosis, congestive heart failure, renal failure, myocardial and vascular wall hypertrophy, systemic hypertension, pulmonary hypertension, and subarachnoid hemorrhage. Specific and nonspecific receptor antagonists and ECE inhibitors that have been developed interfere with endothelin’s function. Many available cardiovascular therapeutic agents, such as angiotensin-converting-enzyme inhibitors, calcium-entry blocking drugs, and nitroglycerin, also may interfere with endothelin release or may modify its activity. The endothelin antagonists have great potential as agents for use in the treatment of a wide spectrum of disease entities and as biologic probes for understanding the actions of endothelin in human beings.

Endothelin receptor antagonists

Sophie Motte, Kathleen McEntee, Robert Naeije
Pharmacology & Therapeutics 110 (2006) 386 – 414
http://dx.doi.org:/10.1016/j.pharmthera.2005.08.012

Endothelin receptor antagonists (ERAs) have been developed to block the effects of endothelin-1 (ET-1) in a variety of cardiovascular conditions. ET-1 is a powerful vasoconstrictor with mitogenic or co-mitogenic properties, which acts through the stimulation of 2 subtypes of receptors [endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) receptors]. Endogenous ET-1 is involved in a variety of conditions including systemic and pulmonary hypertension (PH), congestive heart failure (CHF), vascular remodeling (restenosis, atherosclerosis), renal failure, cancer, and cerebrovascular disease. The first dual ETA/ETB receptor blocker, bosentan, has already been approved by the Food and Drug Administration for the treatment of pulmonary arterial hypertension (PAH). Trials of endothelin receptor antagonists in heart failure have been completed with mixed results so far. Studies are ongoing on the effects of selective ETA antagonists or dual ETA/ETB antagonists in lung fibrosis, cancer, and subarachnoid hemorrhage. While non-peptidic ET-1 receptor antagonists suitable for oral intake with excellent bioavailability have become available, proven efficacy is limited to pulmonary hypertension, but it is possible that these agents might find a place in the treatment of several cardiovascular and non-cardiovascular diseases in the coming future.

Proposed mechanism by which ET-1 triggers vasoconstriction and vascular remodeling. Activation of G-protein-coupled endothelin receptors leads to stimulation of phospholipase C (PLC) which hydrolyses phosphatidyl inositol  biphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG). DAG opens receptor-operated Ca++ channels (ROC) while IP3 induces Ca++ mobilization from the sarcoplasmic reticulum (SR) and opens store-operated Ca++ channels (SOC) directly or indirectly by store depletion to further increase cytosolic Ca++. This Ca++ increase may also trigger Ca++ release from the SR through ryanodine receptors. Depolarization induced by the opening of non-selective cationic channels (NSCC) via ET-1 and Ca++-activated Cl[1] channels as well as by the inhibition of voltage-gated K+ channels (Kv), opens voltage-dependent Ca++ channels (VDCC) to further increase the Ca++ entry across the plasma membrane. The cytosolic Ca++ increase may also activate Na/H exchangers resulting in alkalinization of the cells and promoting Ca++ influx by activating the Na/Ca exchanger. In addition, the elevated cytosolic Ca++ concentrations and DAG activate the protein kinase C and thus promote cell cycle progression by the Ca++/calmodulin complex (Ca++/CaM) and induction of proto-oncogenes. The intracellular signaling cascade induced by activation of ETB receptor is similar to the ETA receptor one, in stimulating the activation of PLC, generating IP3 and DAG and mobilizing of calcium. However, the PLA2 is also activated via ETB receptors to release prostaglandins (PG) and thromboxane A2 (TXA2).

Endothelin-1 increases isoprenaline-enhanced cyclic AMP levels in cerebral cortex

Marıa J. Perez-Alvareza, MC Calcerrada, F Hernandez, RE Catalan, AM Martınez
Regulatory Peptides 88 (2000) 41–46  PII: S0167-0115(99)00118-4

We examined the effect of ET-1 on cyclic AMP levels in rat cerebral cortex. The peptide caused a concentration-dependent increase of [3 H] cyclic AMP accumulation after 10 min of treatment. This effect was due to adenosine accumulation since it was inhibited by the treatment with adenosine deaminase. ET-1, apart from being able to increase cyclic AMP, also potentiated the cyclic AMP generated by isoprenaline in the presence of adenosine deaminase. Experiments performed in the presence of BQ-123 or BQ-788, specific ETA or ETB receptor antagonists respectively indicated that ET was the receptor involved. This effect was dependent on extracellular and B intracellular calcium concentration. These findings suggest that ET-1 plays a modulatory role in cyclic AMP generation systems in cerebral cortex.

Endothelins And Asthma

Roy G. Goldie and Peter J. Henry
Life Sciences I999; 65(1), pp. I-15, PI1 SOO24-3205(98)00614-6

In the decade since endothelin-1 (ET-l) and related endogenous peptides were first identified as vascular endothelium-derived spasmogens, with potential pathophysiological roles in vascular diseases, there has been a significant accumulation of evidence pointing to mediator roles in obstructive respiratory diseases such as asthma. Critical pieces of evidence for this concept include the fact that ET-l is an extremely potent spasmogen in human and animal airway smooth muscle and that it is synthesised in and released from the bronchial epithelium. Importantly, symptomatic asthma involves a marked enhancement of these processes, whereas asthmatics treated with anti-inflammatory glucocorticoids exhibit reductions in these previously elevated indices. Despite this profile, a causal link between ET-l and asthma has not been definitively established. This review attempts to bring together some of the evidence suggesting the potential mediator roles for ET-l in this disease.

Endothelial Cell Peroxisome Proliferator–Activated Receptor ϒ Reduces Endotoxemic Pulmonary Inflammation and Injury

Aravind T. Reddy, SP Lakshmi, JM Kleinhenz, RL Sutliff, CM Hart, and R. Reddy
J Immunol 2012; 189:5411-5420
http://www.jimmunol.org/content/189/11/5411

Bacterial endotoxin (LPS)-mediated sepsis involves severe, dysregulated inflammation that injures the lungs and other organs Bacterial endotoxin (LPS)-mediated sepsis involves severe, dysregulated inflammation that injures the lungs and other organs, often fatally. Vascular endothelial cells are both key mediators and targets of LPS-induced inflammatory responses. The nuclear hormone receptor peroxisome proliferator–activated receptor ϒ (PPARϒ) exerts anti-inflammatory actions in various cells, but it is unknown whether it modulates inflammation through actions within endothelial cells. To determine whether PPARϒ acts within endothelial cells to diminish endotoxemic lung inflammation and injury, we measured inflammatory responses and mediators in mice with endothelial-targeted deletion of PPARϒ. Endothelial cell PPARϒ (ePPARϒ) knockout exacerbated LPS-induced pulmonary inflammation and injury as shown by several measures, including infiltration of inflammatory cells, edema, and production of reactive oxygen species and proinflammatory cytokines, along with upregulation of the LPS receptor TLR4 in lung tissue and increased activation of its downstream signaling pathways. In isolated LPS-stimulated endothelial cells in vitro, absence of PPARϒ enhanced the production of numerous inflammatory markers. We hypothesized that the observed in vivo activity of the ligand-activated ePPARϒ may arise, in part, from nitrated fatty acids (NFAs), a novel class of endogenous PPARϒ ligands.
Supporting this idea, we found that treating isolated endothelial cells with physiologically relevant concentrations of the endogenous NFA 10-nitro-oleate reduced LPS-induced expression of a wide range of inflammatory markers in the presence of PPARϒ, but not in its absence, and also inhibited neutrophil mobility in a PPARϒ-dependent manner. Our results demonstrate a key protective role of ePPARϒ against endotoxemic injury and a potential ePPARϒ-mediated anti-inflammatory role for NFAs.

Endothelins in health and disease

Rahman Shah
European Journal of Internal Medicine 18 (2007) 272–282
http://dx.doi.org:/10.1016/j.ejim.2007.04.002

Endothelins are powerful vasoconstrictor peptides that also play numerous other roles. The endothelin (ET) family consists of three peptides produced by a variety of tissues. Endothelin-1 (ET-1) is the principal isoform produced by the endothelium in the human cardiovascular system, and it exerts its actions through binding to specific receptors, the so-called type A (ETA) and type B (ETB) receptors. ET-1 is primarily a locally acting paracrine substance that appears to contribute to the maintenance of basal vascular tone. It is also activated in several diseases, including congestive heart failure, arterial hypertension, atherosclerosis, endothelial dysfunction, coronary artery diseases, renal failure, cerebrovascular disease, pulmonary arterial hypertension, and sepsis. Thus, ET-1 antagonists are promising new agents. They have been shown to be effective in the management of primary pulmonary hypertension, but disappointing in heart failure. Clinical trials are needed to determine whether manipulation of the ET system will be beneficial in other diseases.

The production of ET receptors is affected by several factors. Hypoxia, cyclosporine, epidermal growth factor, basic fibroblast growth factor, cyclic AMP, and estrogen upregulate ETA receptors in some tissues, and C-type natriuretic hormone, angiotensin II, and perhaps basic fibroblast growth factor up-regulate ETB receptors. In contrast, the endothelins, angiotensin II, platelet-derived growth factor, and transforming growth factor down-regulate ETA receptors, whereas cyclic AMP and catecholamines down-regulate ETB receptors.

The ETA receptor contains 427 amino acids and binds with the following affinity: ET-1N>T-2>ET-3. It is predominantly expressed in vascular smooth muscle cells and cardiac myocytes. Its interaction with ET-1 results in vasoconstriction and cell proliferation. In contrast, the ETB receptor contains 442 amino acids and binds all endothelins with equal affinity. It is predominantly expressed on vascular endothelial cells and is linked to an inhibitory G protein. Activation of ETB receptors stimulates the release of NO and prostacyclin, prevents apoptosis, and inhibits ECE-1 expression in endothelial cells. ETB receptors also mediate the pulmonary clearance of circulating ET-1 and the re-uptake of ET-1 by endothelial cells.

All three endothelins cause transient endothelium dependent vasodilatation before the development of constriction, though this is most apparent for ET-1. Endothelins induce vasodilatation via the endothelial cell ETB receptors through generation of endothelium-derived dilator substances (Fig. 3), including nitric oxide (NO), which perhaps acts by physiologically antagonizing ETA receptor mediated vasoconstriction. The transient early vasodilator actions of the endothelins are attenuated by NO synthase inhibitors.  Additionally, ET-1 increases generation of prostacyclin by cultured endothelial cells, whereas cyclo-oxygenase inhibitors potentiate ET-1-induced constriction, suggesting that vasodilator prostaglandins play a similar modulatory role.

It has been proposed that ET-1 can affect vascular tone indirectly through its effect on the sympathetic nervous system, and it has been shown that that ET-1 may increase peripheral sympathetic activity through postsynaptic potentiation of the effects of norepinephrine. While in vitro low concentrations of ET-1 potentiate the effects of other vasoconstrictor hormones, including norepinephrine and serotonin, these findings have not been confirmed in vivo in the forearm resistance bed of healthy subjects.  In addition to its action on vascular vasomotion, ET-1 is thought to be a mediator in the vascular remodeling process. It seems that ET-1 interactions with the renin–angiotensin–aldosterone system play a significant role in this remodeling process.

Vascular actions of endothelin-1

Vascular actions of endothelin-1

Vascular actions of endothelin-1. Modified from – Galie N, Manes A, Branzi A; The endothelin system in pulmonary arterial hypertension. Cardiovasc Res 2004;61:227–37.

ET-1 appears to have a diverse role as a modulator of vascular tone and growth and as a mediator in many cardiovascular and non-cardiovascular diseases. To date, no disease entity, however, has been attributed solely to an abnormality in ET-1. Yet, ET-1 receptor antagonists have been studied in clinical trials involving a wide spectrum of cardiovascular diseases, though the only proven efficacy has been in patients with PAH.

Learning points

  • Endothelins are powerful vasoconstrictors and major regulators of vascular tone.
  • The endothelin (ET) family consists of three peptides (ET-1 ∼60%, ET-2 ∼30%, and ET-3 ∼10%) produced by a variety of tissues.
  • ET-1 is the principal isoform produced by the endothelium in the human cardiovascular system and appears to be foremost a locally acting paracrine substance rather than a circulating endocrine hormone.
  • Several human studies suggest that circulating ET-1 levels, which are elevated in heart failure and pulmonary hypertension, correlate with the prognosis of the disease.
  • ET-1 antagonists have been shown to be effective in the management of primary pulmonary hypertension, but disappointing in heart failure.
  • Clinical trials are needed to investigate the role of ET-1 receptor antagonists for other conditions, as ET-1 levels have been shown to be elevated in arterial hypertension, atherosclerosis, endothelial dysfunction, coronary artery disease, renal failure, cerebrovascular disease, and sepsis.

In Vitro Stability and Intestinal Absorption Characteristics of Hexapeptide Endothelin Receptor Antagonists

Hyo-kyung Han, BH Stewart, AM Doherty, WL Cody and GL Amidon
Life Sciences. I998; 63(18), pp. 1599-1609. PI1 SOO24-3205(98)00429-9

Endothelins are potent vasoconstrictor peptides which have a wide range of tissue distribution and three receptor subtypes (ETA ETB and ETC). Among the linear hexapeptide ETA / ETB receptor antagonists, PD 145065 (Ac-D-Bhg-L-Leu-L-Asp-L-Ile-L-Ile-L-Trp,  Bhg = (10,ll -dihydro-5H-dibenzo[a,d]cyclohepten-5-yl)-Gly) and PD 156252 (Ac-o-Bhg-L-Leu-L-Asp-L-Ile-(N-methyl)-L-Ile-L-Trp) were selected to evaluate the metabolic stability and intestinal absorption in the absence and/or in the presence of protease inhibitors. In vitro stability of both compounds was investigated in fresh plasma, lumenal perfusate, intestinal and liver homogenates. PD 156252 was more stable than PD 145065 in intestinal tissue homogenate (63.4% vs. 20.5% remaining) and liver homogenate (74.4% vs. 35.5 % remaining), while both compounds showed relatively good stability in the fresh plasma (94.5% vs. 86.7% remaining) and lumenal perfusate (85.8% vs. 72.3% remaining). The effect of protease inhibitors on the degradation of PD 145065 and PD 156252 was also investigated. Amastatin, thiorphan, chymostatin and the mixture of these three inhibitors were effective in reducing the degradation of both compounds. The pharmacokinetic parameters of PD 156252, calculated by using a non-compartmental model, were 6.95 min (terminal half-life), 191 mL (Vss), and 25.5 mL/min (Cltot) after intravenous administration in rats. The intestinal absorption of PD 156252 in rats was evaluated in the absence and/or in the presence of protease inhibitors. The results indicate that the major elimination pathway of PD 156252 appears to be the biliary excretion and protease inhibitors increase the intestinal absorption of PD 156252 through increasing metabolic stability.

Inhibitory and facilitatory presynaptic effects of endothelin on sympathetic cotransmission in the rat isolated tail artery

Violeta N. Mutafova-Yambolieva & David P. Westfall
British Journal of Pharmacology (1998) 123, 136 – 142

1 The present study was undertaken to determine the modulatory effects of the endothelin peptides on the neurogenically-induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5′-triphosphate (ATP) from the sympathetic nerves of endothelium-free segments of the rat isolated tail artery. The electrical field stimulation (EFS, 8 Hz, 0.5 ms, 3 min) evoked over¯ow of NA and ATP, in the absence of endothelins, was 0.035+0.002 pmol mg71 tissue and 0.026+0.002 pmol mg71 tissue, respectively.

2 Endothelin-1 (ET-1; 1 ± 30 nM) significantly reduced the EFS evoked overflow of both NA and ATP.  The maximum inhibitory effect was produced by a peptide concentration of 10 nM, the amount of NA overflow being 0.020+0.002 pmol mg71 and that of ATP overflow 0.015+0.001 pmol mg71. Higher peptide concentrations (100 and 300 nM) reversed the EFS-evoked overflow of NA to control levels and that of ATP to above control levels. The inhibitory effect of ET-1 (10 nM) was resistant to the selective ETA receptor antagonist cyclo-D-Trp-D-Asp(ONa)-Pro-D-Val-Leu (BQ-123) but was prevented by ETB receptor desensitization with sarafotoxin S6c (StxS6c) or by ETB receptor blockade with N, cis-2,6-dimethyl-piperidinocarbonyl-L-gmethylleucyl-D-1-methoxycarbonyl-tryptophanyl-D-norleucine (BQ-788).

3 StxS6c, upon acute application, exerted a dual effect on transmitter release. At concentrations of 0.001 ± 0.3 nM the peptide significantly reduced the EFS-evoked NA overflow, whereas at concentrations of 1 ± 10 nM it caused a significant increase in the evoked overflow of both ATP and NA. Both the maximum inhibitory effect of StxS6c at a concentration of 0.003 nM approximately 85% reduction of NA overflow and 40% of ATP overflow) and the maximum facilitatory effect of the peptide at a concentration of 3 nM (approximately 400% increase of ATP overflow and 200% of NA overflow) were completely antagonized by either BQ-788 or by StxS6c-induced ETB receptor desensitization.

4 ET-3 (10 ± 100 nM) did not a€ect the EFS evoked overflow of either ATP or NA, but at a concentration of 300 nM significantly potentiated the release of both transmitters (0.118+ 0.02 pmol mg71 tissue ATP overflow and .077+0.004 pmol mg71 NA overflow). This effect was prevented either by BQ-123 or by BQ-788.

5 In summary, the endothelin peptides exerted both facilitatory and inhibitory effects on the neurogenically-induced release of the sympathetic cotransmitters ATP and NA in the rat tail artery. Both transmitters were modulated in parallel indicating that the endothelins do not differentially modulate the release of NA and ATP in this tissue.

Involvement of the central adrenomedullin peptides in the baroreflex

Meghan M. Taylo, Cynthia A. Keown, Willis K. Samson
Regulatory Peptides 112 (2003) 87– 93
http://dx.doi.org:/10.1016/S0167-0115(03)00026-0

The peptides derived from post-translational processing of preproadreno-medullin are produced in and act on areas of the autonomic nervous system important for blood pressure regulation. We examined the role of endogenous, brain-derived adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) in the central nervous system arm of the baroreflex by using passive immunoneutralization to block the actions of the endogenous peptides. Our results indicate that the preproadrenomedullin-derived peptides do not play a role in sensing changes in blood pressure (baroreflex sensitivity), but the adrenomedullin peptides do regulate the speed with which an animal returns to a normal, stable blood pressure. These findings suggest that endogenous, brain-derived AM and PAMP participate in the regulation of autonomic activity in response to baroreceptor activation and inactivation.

Pharmacological characterization of cardiovascular responses induced by endothelin-1 in the perfused rat heart

Keiji Kusumoto, A Fujiwara, S Ikeda, T Watanabe, M Fujino
Eur J Pharmacology 296 (1996) 65-74 SSDI 0014-2999(95)00680-X

The effects of the endothelin receptor antagonist TAK-044 (cyclo[D-α-aspartyl-3-[(4-phenylpiperazin-l-yl)carbonyl]-L-alanyl-L-α-aspartyl-D-2-(2-thienyl)-glycyl-L-leucyl-D-tryptophyl] disodium salt) and BQ-123 (cyclo[D-Asp-Pro-D-VaI-Leu-D-Trp]) were studied in the rat heart to characterize the receptor subtypes responsible for the cardiovascular actions of endothelin-1. Endothelin-1 induced a transient decrease and subsequent increase in perfusion pressure in perfused rat hearts, and increased left ventricular developed pressure. TAK-044 diminished these endothelin-l-induced responses (100 pmol/heart) with IC50 values of 140, 57 and 1.3 nM, respectively. BQ-123 (1-30/µM) partially inhibited the endothelin-l-induced hypertension (30-40%) in the rat heart, and failed to inhibit the hypotension. The positive inotropic effect of endothelin-1 was abolished by BQ-123. Neither indomethacin (10/µM) nor N’°-nitro-L-arginine methyl ester (100/pM) attenuated the  endothelin-l-induced hypotension. TAK-044 and BQ-123 attenuated the positive inotropic effect of endothelin-1 in rat papillary muscles. In rat cardiac membrane fractions, TAK-044 and BQ-123 inhibited [125I]endothelin-1 binding to endothelin ET A receptors with IC50 values of 0.39 + 0.6 and 36 + 9 nM, respectively, whereas only TAK-044 potently blocked the endothelin ET B receptor subtype (IC50 value: 370 + 180 nM). These results suggest that endothelin-1 modulates cardiovascular functions in the rat heart by activating both endothelin ET A and endothelin ET B receptors, all of which are sensitive to TAK-044.

Molecular Pharmacology and Pathophysiological Significance of Endothelin

Katsutoshi Goto, Hiroshi Hama and Yoshitoshi Kasuya
Jp J Pharmacol 1996; 72: 261-290

Since the discovery of the most potent vasoconstrictor peptide, endothelin, in 1988, explosive investigations have rapidly clarified much of the basic pharmacological, biochemical and molecular biological features of endothelin, including the presence and structure of isopeptides and their genes (endothelin- 1, -2 and -3), regulation of gene expression, intracellular processing, specific endothelia converting enzyme (ECE), receptor subtypes (ETA and ETB), intracellular signal transduction following receptor activation, etc. ECE was recently cloned, and its structure was shown to be a single transmembrane protein with a short intracellular N-terminal and a long extracellular C-terminal that contains the catalytic domain and numerous N-glycosylation sites. In addition to acute contractile or secretory actions, endothelin has been shown to exert long-term proliferative actions on many cell types. In this case, intracellular signal transduction appears to converge to activation of mitogen-activated protein kinase. As a recent dramatic advance, a number of non-peptide and orally active receptor antagonists have been developed. They, as well as current peptide antagonists, markedly accelerated the pace of investigations into the true pathophysiological roles of endogenous endothelin-1 in mature animals.

The discovery of endothelin in 1988 soon triggered explosive investigations of a worldwide scale, presumably due to its unusual characteristics; i.e., marked potency and long-lasting pressor actions. As a result, most of the basic problems concerned with the science of endothelin have rapidly been solved; e.g., features and regulations of the expression of endothelin genes,  biosynthetic pathways including characterization and cloning of endothelin converting enzyme, pharmacological, biochemical and molecular-biological identification of endothelin receptor subtypes, intracellular signal transduction following receptor activation, and discovery of various receptor agonists and antagonists. In addition to its potent cardiovascular actions, endothelin-1 shows a wide variety of biological effects, including contraction of nonvascular smooth muscle (intestinal, tracheal, broncheal, mesangial, bladder, uterine and prostatic smooth muscle), stimulation of neuropeptides, pituitary hormone and atrial natriuretic peptide release and aldosterone biosynthesis, modulation of neurotransmitter release, and increase of bone resorption. Furthermore, endothelin-1 has mitogenic properties and causes proliferation and hypertrophy of a number of cell types, including vascular smooth muscle cells, cardiac myocytes, mesangial cells, bronchial smooth muscle cells and fibroblasts. Endothelin-1 also induces the expression of several protooncogenes (c fos, C -Jun, c-myc, etc.).

These actions, whereby endothelin- 1 might influence the development of cellular hypertrophy/hyperplasia, are of potential significance in pathophysiological conditions associated with long-term changes in cardiovascular tissues, e.g., hypertension, myocardial infarction, chronic heart failure, vascular restenosis following balloon angioplasty, and atherosclerosis. These pathophysiological conditions are usually associated with increased plasma levels of endothelin-1, although the correlation is relatively poor. Nevertheless, a considerable increase in the tissue content of endothelin-1 has been gradually uncovered in many cases of these conditions. Even if the concentration of endothelin-1 at the cell surface is not high enough to induce contraction, it is well known that subthreshold concentrations of endothelin will enhance or potentiate the contraction produced by other vasoconstrictors (e.g., norepinephrine, serotonin, angiotensin II), indicating the existence of cross-talk among various vasoactive substances. Another important cross-talk among these substances may be mutual enhancement or inhibition of their expression in various tissues. In addition to these interactions, the true physiological and/or pathophysiological roles of each of the endothelin family peptide and receptor subtypes remain to be investigated.

Hydrogen Sulfide and Endothelium-Dependent Vasorelaxation

Jerzy Bełtowski, and Anna Jamroz-Wiśniewska
Molecules 2014, 19, 21183-21199; http://dx.doi.org:/10.3390/molecules191221183

In addition to nitric oxide and carbon monoxide, hydrogen sulfide (H2S), synthesized enzymatically from L-cysteine or L-homocysteine, is the third gasotransmitter in mammals. Endogenous H2S is involved in the regulation of many physiological processes, including vascular tone. Although initially it was suggested that in the vascular wall H2S is synthesized only by smooth muscle cells and relaxes them by activating ATP-sensitive potassium channels, more recent studies indicate that H2S is synthesized in endothelial cells as well. Endothelial H2S production is stimulated by many factors, including acetylcholine, shear stress, adipose tissue hormone leptin, estrogens and plant flavonoids. In some vascular preparations H2S plays a role of endothelium-derived hyperpolarizing factor by activating small and intermediate-conductance calcium-activated potassium channels. Endothelial H2S signaling is up-regulated in some pathologies, such as obesity and cerebral ischemia-reperfusion. In addition, H2S activates endothelial NO synthase and inhibits cGMP degradation by phosphodiesterase thus potentiating the effect of NO-cGMP pathway. Moreover, H2S-derived polysulfides directly activate protein kinase G. Finally, H2S interacts with NO to form nitroxyl (HNO)—a potent vasorelaxant. H2S appears to play an important and multidimensional role in endothelium-dependent vasorelaxation.

GPCR modulation by RAMPs

Debbie L. Hay, David R. Poyner, Patrick M. Sexton
Pharmacology & Therapeutics 109 (2006) 173 – 197
http://dx.doi.org:/10.1016/j.pharmthera.2005.06.015

Our conceptual understanding of the molecular architecture of G-protein coupled receptors (GPCRs) has transformed over the last decade. Once considered as largely independent functional units (aside from their interaction with the G-protein itself), it is now clear that a single GPCR is but part of a multifaceted signaling complex, each component providing an additional layer of sophistication. Receptor activity modifying proteins (RAMPs) provide a notable example of proteins that interact with GPCRs to modify their function. They act as pharmacological switches, modifying GPCR pharmacology for a particular subset of receptors. However, there is accumulating evidence that these ubiquitous proteins have a broader role, regulating signaling and receptor trafficking. This article aims to provide the reader with a comprehensive appraisal of RAMP literature and perhaps some insight into
the impact that their discovery has had on those who study GPCRs.

RAMPs were first identified during attempts to expression clone a receptor for the neuropeptide calcitonin gene related peptide (CGRP; McLatchie et al., 1998). Historical evidence had suggested that CGRP acted through a GPCR, as its binding had proven sensitive to GTP analogues and stimulation of various tissues and cells led to the accumulation of cAMP, suggesting activation of a Gs-coupled GPCR. However, attempts to clone such a receptor proved difficult. A putative canine CGRP receptor, RDC-1, was identified in 1995, but the original findings have not been replicated and current IUPHAR guidelines do not consider this receptor a genuine CGRP receptor (Kapas & Clark, 1995; Poyner et al., 2002). Shortly afterward, a further orphan receptor (CL, a close homologue of the calcitonin receptor) was shown to be activated by CGRP when transfected into HEK293 cells (Aiyar et al., 1996). This finding posed something of a conundrum since earlier attempts to examine the function of this receptor (or its rat homologue) in Cos 7 cells had not given positive results with CGRP.
Given the apparent functionality of the human CL receptor in HEK293 cells, the rat homologue was also transfected into this cell type and now responded to CGRP (Han et al., 1997). The authors speculated that there was a factor present in HEK293 cells that conferred high affinity for CGRP on the receptor.

In 1998, McLatchie and colleagues confirmed this speculation and provided new insights into the way that GPCRs and their pharmacology can be regulated (McLatchie et al., 1998). It was discovered that a novel family of single transmembrane domain proteins, termed RAMPs, was required for functional expression of CL at the cell surface, explaining why it had been so difficult to observe CGRP binding or function when CL was transfected into cells lacking RAMP expression (Fluhmann et al., 1995; Han et al., 1997; McLatchie et al., 1998). RAMPs were first identified from a library derived from SK-N-MC cells, cells known to express CGRP receptors. An expression-cloning strategy was utilized, whereby an SK-N-MC cDNA library was transcribed and the corresponding cRNA was used for injection into Xenopus oocytes. Cystic
fibrosis transmembrane regulator chloride conductance, a reporter for cAMP formation, was strongly potentiated by a single cRNA pool (in the presence of CGRP). Subsequently, a single cDNA encoding a 148-amino-acid protein comprising RAMP1 was isolated. The structure of the protein was unexpected, as it was not a GPCR and it did not respond to CGRP in mammalian cells. Thus, it was postulated that RAMP1 might potentiate CGRP receptors. A CL/RAMP1 co-transfection experiment supported this hypothesis.

CGRP/AM on the outside of the cell and did not simply act as anchoring/chaperone proteins for CL. RAMPs therefore provide a novel mechanism for modulating receptor–ligand specificity. The unique pharmacological profiles supported by RAMPs are discussed in later sections.

Fig. (not shown).  CGRP1 receptor-specific small molecule antagonists. The small molecule antagonist BIBN4096 BS (brown) is a specific antagonist of the CGRP1 receptor, acting at the interface between RAMP1 and the CL receptor to inhibit CGRP action. At least part of the binding affinity for BIBN4096 BS arises from interaction with Trp74 (red) of RAMP1. In contrast, antagonists that bind principally to the CL component of the complex will not discriminate between different CL/RAMP complexes.

The classic function attributed to RAMPs is their ability to switch the pharmacology of CL, thus providing a novel mechanism for modulating receptor specificity. Thus, the CL/RAMP1 complex is a high affinity CGRP receptor, but in the presence of RAMP2, CL specificity is radically altered, the related peptide AM being recognized with the highest affinity and the affinity for CGRP being reduced ¨100-fold. While AM is the highest affinity peptide, CGRP is recognized with moderate, rather than low affinity. Indeed, depending on the species and the form of CGRP (h vs. a), the separation between the 2 peptides can be as little as 10-fold (Hay et al., 2003a). This may particularly be true if receptor components of mixed species are used. The detailed pharmacology of the CGRP and AM receptors formed by RAMP interaction with CL has recently been reviewed (Born et al., 2002; Poyner et al., 2002; Hay et al., 2004; Kuwasako et al., 2004).

Fig. (not shown). The broadening spectrum of RAMP–receptor interactions. RAMPs can interact with multiple receptor partners. All RAMPs interact with the calcitonin receptor-like receptor (CL-R), the calcitonin receptor (CTR), and the VPAC1 receptor, while the glucagon and PTH1 receptors interact with RAMP2, the PTH2 receptor with RAMP3, and the calcium sensing receptor (CalS-R) with RAMP1 or RAMP3. The consequence of RAMP interaction varies. For the CL and CalS receptors, RAMPs play a chaperone role, allowing cell surface expression. For the CL and calcitonin receptors, RAMP interaction leads to novel receptor binding phenotypes . There is also evidence that RAMP interaction will modify signaling, and this has been seen for the VPAC1–RAMP2 heterodimer and for calcitonin receptor/RAMP complexes. In many instances, however, the consequence of RAMP interaction has yet to be defined.

Overall, the distribution data presented so far are supportive of the hypothesis that RAMP and CL or calcitonin receptor combinations are able to account for the observed CGRP, AM, and AMY pharmacology. A salient point for CGRP receptors relates to the cerebellum, where the lack of CL mRNA in some studies despite abundant CGRP binding has prompted speculation of alternative CGRP receptors (Oliver et al., 2001; Chauhan et al., 2003). Nevertheless, this apparent lack is study dependent and CL has been identified in cerebellum in other studies.

Some consideration has been given to the potential role that RAMPs may have in modifying receptor behaviors other than ligand binding pharmacology. An additional functional consequence might be that of alteration of receptor signaling characteristics.

While there is currently little evidence for signaling modifications of CL-based receptors in association with RAMPs, a completely different paradigm is evident for the VPAC1 receptor. This receptor has strong interactions with all 3 RAMPs, but its pharmacology, in terms of agonist binding, does not appear to be modified by their presence. On the other hand, there was a clear functional consequence of RAMP2 overexpression with the VPAC1 receptor where PI hydrolysis was specifically augmented relative to cAMP, which did not change. The potency of the response (EC50 of vasoactive intestinal peptide) was not altered, but the maximal PI hydrolysis response was elevated in the presence of RAMP2 . It has been suggested that this may reflect a change in compartmentalization of the receptor signaling complex. Such augmentation was not evident for the interaction of the VPAC1 receptor with RAMP1 or RAMP3; in these cases, the outcome of heterodimerization may be more subtle or involve the modification of different receptor parameters such as trafficking.

RAMPs transformed our understanding of how receptor pharmacology can be modulated and provided a novel mechanism for generating receptor subtypes within a subset of family B GPCRs. Their role has now broadened and they have been shown to interact with several other family B GPCRs, in 1 case modifying signaling parameters. There is now evidence to suggest that their interactions also reach into family C, and possibly family A, GPCRs, indicating that their function may not be restricted to modulation of a highly specific subset of receptors. Indeed, many aspects of RAMP function remain poorly understood, and the full extent of their action remains to be explored.

Receptor activity modifying proteins

Patrick M. Sexton, Anthony Albiston, Maria Morfis, Nanda Tilakaratne
Cellular Signalling 13 (2001) 73-83  PII: S0898-6568(00)00143-1

Our understanding of G protein-coupled receptor (GPCR) function has recently expanded to encompass novel protein interactions that underlie both cell-surface receptor expression and the exhibited phenotype. The most notable examples are those involving receptor activity modifying proteins (RAMPs). RAMP association with the calcitonin (CT) receptor-like receptor (CRLR) traffics this receptor to the cell surface where individual RAMPs dictate the expression of unique phenotypes. A similar function has been ascribed to RAMP interaction with the CT receptor (CTR) gene product. This review examines
our current state of knowledge of the mechanisms underlying RAMP function.

It is now evident that RAMPs can interact with receptors other than CRLR. Expression of amylin receptor phenotypes requires the coexpression of
RAMPs with the CTR gene product. However, as seen in CRLR, the phenotype engendered by individual RAMPs was distinct. In COS-7 or rabbit aortic endothelial cells (RAECs), RAMP1 and RAMP3 induced amylin receptors that differ in their affinity for CGRP, while RAMP2 was relatively ineffective in inducing amylin receptor phenotype. RAMP2 can also induce an amylin receptor phenotype, which is distinct from either the RAMP1- or RAMP3-induced receptors. However, the efficacy of RAMP2 was highly dependent upon the cellular background and the isoform of CTR used in the study.

In humans, the major CTR variants differ by the presence or absence of a 16 amino acid insert in the first intracellular domain, with the insert negative isoform (hCTRI1ÿ) being the most commonly expressed form and the variant used for initial studies with RAMPs. Unlike hCTRI1ÿ, cotransfection of the hCTRI1+ variant with any of the RAMPs into COS-7 cells caused strong induction of amylin receptor phenotype. The hCTR isoforms differ in their ability to activate signaling pathways (presumably due to an effect on G protein coupling) and to internalize in response to agonist treatment, which may suggest a role for G proteins in the ability of RAMPs to alter receptor phenotype.

There are at least three potential consequences of RAMP interaction with its associating receptors. The first is trafficking of receptor protein from an intracellular compartment to the cell surface. The second is an alteration in
the terminal glycosylation of the receptor, and the third is alteration of receptor phenotype, presumably through a direct or indirect effect on the ligand-binding site.

potential actions of RAMPs

potential actions of RAMPs

Schematic diagram illustrating potential actions of RAMPs. (A) RAMPs facilitate the trafficking of CRLR from an intracellular compartment to the cell surface. (B) RAMP1 (but not RAMP2 or RAMP3) modifies the terminal glycosylation
of CRLR. (C) The cell surface RAMP1±CRLR complex is a Type 1 CGRP receptor, which displays a 1:1 stoichiometry. (D,E) Cell surface RAMP2±CRLR and  RAMP3±CRLR complexes are adrenomedullin receptors. (F,G) For at least RAMP1 and RAMP3, RAMPs form stable homodimers, although the function
of these complexes is unknown. (H) Unlike CRLR, the CTR gene product is trafficked to the cell surface in the absence of RAMPs, where it displays classical CTR phenotype. (I,J) RAMP1± and RAMP3±CTR complexes form distinct amylin receptors. RAMP2 can also generate a separate amylin receptor phenotype (not illustrated). (C ±E,I,J) RAMPs are trafficked with either receptor to the plasma membrane. (K) For all three RAMP±CRLR complexes, agonist treatment causes clathrin-mediated internalization of both CRLR and RAMP.
(L) The majority of the internalized complex is targeted to the lysosomal-degradation pathway.

The data from Zumpe et al. suggest that RAMP2 interacts more weakly with the hCTRI1ÿ than RAMP1, and that the affinity of this interaction derives principally from the transmembrane domain/C-terminus (Ct) of the RAMPs. As RAMP3 induces an amylin receptor phenotype in COS-7 cells where RAMP2 is relatively weak, it is inferred that RAMP3 interaction with the hCTRI1ÿ is probably greater than that of RAMP2. Nonetheless, this has not been examined empirically. Given the recent data suggesting a potential role for G protein coupling in expression of RAMP-induced phenotype, it is also possible that the strength of RAMP interaction is, at least partially, dictated by receptor-G protein or RAMP-G protein interaction.

The discovery of RAMPs has led to a greater understanding of the nature of receptor diversity. However, although much progress has been made into elucidating the molecular mechanism of RAMP action, emerging data continue to open up new areas for investigation. These include identification of other RAMP-interacting receptors, understanding of the role of specific G proteins in RAMP-receptor function and the potential importance of RAMP regulation in disease progression. It also seems likely that the RAMP-receptor interface can provide a useful target for future drug development.

Cardiovascular endothelins: Essential regulators of cardiovascular homeostasis

Friedrich Brunner, C Bras-Silva, AS Cerdeira, AF Leite-Moreira
Pharmacology & Therapeutics 111 (2006) 508 – 531
http://dx.doi.org:/10.1016/j.pharmthera.2005.11.001

The endothelin (ET) system consists of 3 ET isopeptides, several isoforms of activating peptidases, and 2 G-protein-coupled receptors, ETA and ETB, that are linked to multiple signaling pathways. In the cardiovascular system, the components of the ET family are expressed in several tissues, notably the vascular endothelium, smooth muscle cells, and cardiomyocytes. There is general agreement that ETs play important physiological roles in the regulation of normal cardiovascular function, and excessive generation of ET isopeptides has been linked to major cardiovascular pathologies, including hypertension and heart failure. However, several recent clinical trials with ET receptor antagonists were disappointing.

In the present review, the authors take the stance that ETs are mainly and foremost essential regulators of cardiovascular function, hence that antagonizing normal ET actions, even in patients, will potentially do more harm than good. To support this notion, we describe the predominant roles of ETs in blood vessels, which are (indirect) vasodilatation and ET clearance from plasma and interstitial spaces, against the background of the subcellular mechanisms mediating these effects. Furthermore, important roles of ETs in regulating and adapting heart functions to different needs are addressed, including recent progress in understanding the effects of ETs on diastolic function, adaptations to changes in preload, and the interactions between endocardial-derived ET-1 and myocardial pump function. Finally, the potential dangers (and gains) resulting from the suppression of excessive generation or activity of ETs occurring in some cardiovascular pathological states, such as hypertension, myocardial ischemia, and heart failure, are discussed.

Figure (not shown):  Synthesis of ET and its regulation. The release of active ET-1 is controlled via regulation of gene transcription and/or endothelin converting enzyme activity. ET-1 synthesis is stimulated by several factors, of which hypoxia seems to be the most potent in humans (see text). ET-1 formation is down-regulated by activators of the NO/cGMP pathway and other factors.

Figure (not shown): Vascular actions of ET. In healthy blood vessels, the main action of ET-1 is indirect vasodilatation mediated by ETB receptors located on endothelial cells. Their activation generates a Ca2+ signal via PLC that turns on the generation of NO, prostacyclin, adrenomedullin, and other mediators that are powerful relaxants of smooth muscle. On the other hand, binding of ET-1 to ETA receptors located on smooth muscle cells will lead to vascular contraction (physiological effect) and/or wall thickening, inflammation, and tissue remodeling (pathological effects). These latter effects may partly be mediated by vascular ETB2 receptors in certain disease states. Smooth muscle cell signaling involves DAG formation, PKC activation, and extracellular Ca2+ recruited via different cation channels. The specificity of the cellular response resides at the level of G proteins, that is, G-as or G-aq in the case of ETA, G-ai or G-aq for ETB.

signal transduction mechanisms involved in ET-1-mediated positive (left) and negative (right) inotropic effects

signal transduction mechanisms involved in ET-1-mediated positive (left) and negative (right) inotropic effects

Summary of proposed signal transduction mechanisms involved in ET-1-mediated positive (left) and negative (right) inotropic effects. Left: Stimulation of ETA receptors causes Gq protein-directed activation of PLC, formation of IP3 and DAG, and activation of NHE-1. Increased contractile force is the result of (i) Ca2+ release from the sarco(endo)plasmic reticulum, (ii) sensitization of cardiac myofilaments to Ca2+ due to cellular alkalosis, and (iii) increased Ca2+ influx through the NCX operating in reverse mode. The contribution of voltage-gated L-type Ca2+ channels to the systolic Ca2+ transient is unknown, as is the role of myocyte ETB2 receptors. Right: The ET receptor subtypes mediating negative inotropic effects are poorly known. Two main signaling mechanisms involve (i) inhibition of adenylyl cyclase (AC), guided by a G protein, of unknown binding preference, which results in decreased levels of cAMP; (ii) cGMP-mediated activation of phosphatases that dephosphorylate putative targets resulting from cAMP/protein kinase A (PKA) activation. Other kinases like PKC and PKG have also been implicated in accentuated force antagonism.

Adrenomedullin (11–26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla

Kazuo Kitamuraa,*, Eizaburo Matsuia, Jhoji Katoa, Fumi Katoha
Peptides 22 (2001) 1713–1718 PII: S0196-9781(01)00529-0

Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from pheochromocytoma tissue. Both the ring structure and the C-terminal amide structure of AM are essential for its hypotensive activity. We have developed an RIA which recognizes the ring structure of human AM. Using this RIA, we have characterized the molecular form of AM in bovine adrenal medulla. Gel filtration chromatography revealed that three major peaks of immunoreactive AM existed in the adrenal medulla. The peptide corresponding to Mr 1500 Da was further purified to homogeneity. The peptide was determined to be AM (11–26) which has one intramolecular disulfide bond. Amino acid sequences of bovine AM and its precursor were deduced from the analyses of cDNA encoding bovine AM precursor. The synthetic AM (11–26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. The hypertensive activity lasted about one minute, and a dose dependent increase in heart rate was also observed. The present data indicate that AM (11–26) is a major component of immunoreactive AM in bovine adrenal medulla and shows pressor activity.

The pressor effect of AM(11–26) was examined by methods similar to those reported for Neuropeptide Y.

We have established a sensitive RIA system using a monoclonal antibody which recognizes the ring structure of human AM. Human AM antiserum recognized the peptide with high affinity at a final dilution of 1:2,800,000. The half maximal inhibition of radioiodinated ligand binding by human AM was observed at 10 fmol/tube. From 1 to 128 fmol/tube of AM was measurable by this RIA system. The intra- and inter-assay coefficients of variance were less than 6% and 9%, respectively. This RIA had 100% cross-reactivity with human AM(13–31), (1–25), (1–52)Gly and AM(1–52)CONH2, but less than 1% cross-reactivity with rat AM.

Sephadex G-50 gel-filtration of strongly basic peptide extract (SP-III) in bovine adrenal medulla identified three major peaks of immunoreactive AM. One emerged at the identical position of authentic AM, the other two unknown peaks were eluted later at molecular weights estimated to be 3000 and 1500 Da, respectively. The peptide corresponding to Mr 1500 Da was further purified.

The purified peptide (20 pmol) was subjected to a gas phase sequencer, and the amino acid sequence was determined up to the 16th residue, which was found to be C terminus . It was found that the purified peptide was AM (11–26). The structure of AM (11–26) was confirmed by chromatographic comparison with native AM (11–26) as well as a synthetic AM (11–26), which has one intramolecular disulfide bond.

3 clones were isolated, and the clone designated pBAM-2, which harbored the longest insert of 1,438 base, was used for sequencing. The bovine AM cDNA contained a single open reading frame encoding a putative 188 amino acid polypeptide. The first 21-residue peptide is thought to be a signal peptide. The bovine AM propeptide contains three signals of dibasic amino acid sequences, Lys-Arg or Arg-Arg. The first Lys-Arg followed proadrenomedullin N-terminal 20 peptide (PAMP) sequences. AM is located between the second signal of Lys-Arg and the third signal of Arg-Arg. Gly residues, which are donors of C-terminal amide structure of PAMP and AM, are found before the first and third signal of Lys-Arg and Arg-Arg. Bovine AM consists of 52 amino acids and is identical to human AM with exception of four amino acids. Bovine PAMP consists of 20 amino acids and is identical to human PAMP with exception of one amino acid. The present cDNA sequence encoding bovine AM precursor is almost identical to those of the reported AM cDNA sequences from bovine aortic endothelial cells. However, a difference in one amino acid was found in the sequences of signal peptide. In addition, three different residues of nucleotides were found in the noncoding region of cDNA encoding bovine preproadreno-medullin.

AM(11–26) elicited a potent hypertensive effects in unanesthetized rats.
When AM(11–26) at 20 nmol/kg was injected i.v., the maximum increase of mean blood pressure was 50  7.1 mmHg. Similarly, the synthetic AM(11–26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. (Blood pressure increase; F(3, 20 = 13.845, P < 0.0001). Injection of saline did not affects blood pressure and heart rate. The hypertensive activity lasted about 70 s, and a dose dependent increase of heart rate was also observed (Heart rate increase; F(3, 20) = 6.151, P = 0.0039).

We have isolated and characterized bovine AM(11–26) from bovine adrenal medulla as an endogenous peptide. The hallmark biological effects of AM are vasodilation and hypotensive effects in the vascular systems of most species. The mature form of AM has one ring structure formed by an intramolecular disulfide bond and a C terminal amide structure, both of which are essential for the hypotensive and other biological activities of AM. Watanabe et al. reported that the synthetic N-terminal fragment of human AM, AM (1–25)COOH and other related peptides, show vasopressor activity in anesthetized rats. The present purification and characterization of AM(11–26) indicate that the ring structure of AM may function as a biologically active endogenous peptide. The peptide corresponding to Mr 1,500 Da was further purified to homogeneity.

The purified peptide was found to be AM(11–26) which has one intramolecular disulfide bond. The structure of AM(11–26) was confirmed by chromatographic comparison with native AM(11–26) as well as a synthetic specimen which was prepared according to the determined sequence. The structure of bovine AM and related peptides were determined by cDNA analysis encoding bovine AM. Bovine AM consists of 52 amino acids whose sequence is identical to the human sequences with the exception of four amino acids. Furthermore, according to the cDNA analysis and chromatographic comparison of the synthetic AM(11–26) and purified AM, is now determined to be cystine. It should be noted that the structure of bovine AM(11–26) is identical to human AM(11–26).

It is well known that many peptide hormones and neuropeptides are processed from larger, biologically inactive precursors by the specific processing enzyme. It usually recognizes pairs of basic amino acids, processing signals, such as primarily Lys-Arg and Arg-Arg. AM (11–26) is not flanked by such a processing signal, but it was reproducibly observed in bovine adrenal medulla peptide extract. The molar ratio of AM(11–26)/AM was estimated to be 40%. The ratio varied from 5% to 50% according to the individual specimen, but the minor peak corresponding to 1,500 Da was reproducibly observed, suggesting that AM(11–26) is an endogenous peptide. It is likely that AM(11–26) is biosynthesized from AM or AM precursor by a specific enzyme.

In contrast to AM, synthetic bovine AM(11–26) caused potent hypertensive effects in unanesthetized rats. The hypertensive activity of AM(11–26) seems to be comparable to that of AM(1–25) as reported by Watanabe et al.  It was unexpected that AM(11–26) would cause a dose dependent increase of heart rate in unanesthetized rats because vasopressor activity normally causes bradycardia through baroreceptor activation. The hypertensive mechanism is not fully understood, but it has been reported that the vasopressor effect of AM(1–25) might be caused by the release of endogenous catecholamine. We speculate that the released catecholamine counters the baroreceptor function resulting in an increased heart rate and blood pressure. It is possible that AM(11–26) participates in blood pressure control as an endogenous peptide.

A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides.

Tanenao Eto
Peptides 22 (2001) 1693–1711 PII: S0196-9781(01)00513-7

Adrenomedullin (AM), identified from pheochromocytoma and having 52 amino acids, elicits a long-lasting vasodilatation and diuresis. AM is mainly mediated by the intracellular adenylate cyclase coupled with cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) -cyclic guanosine monophosphate (cGMP) pathway through its specific receptor. The calcitonin receptor-like receptor (CLCR) and receptor-activity modifying protein (RAMP) 2 or RAMP3 models have been proposed as the candidate receptor. AM is produced mainly in cardiovascular tissues in response to stimuli such as shear stress and stretch, hormonal factors and cytokines. Recently established AM knockout mice lines revealed that AM is essential for development of vitelline vessels of embryo. Plasma AM levels elevate in cardiovascular diseases such as heart failure, hypertension and septic shock, where AM may play protective roles through its characteristic biological activities. Human AM gene delivery improves hypertension, renal function, cardiac hypertrophy and nephrosclerosis in the hypertensive rats. AM decreases cardiac preload and afterload and improves cardiac contractility and diuresis in patients with heart failure and hypertension. Advances in gene engineering and receptor studies may contribute to further understandings of biological implication and therapeutic availability of AM.

AM acts as a circulating hormone as well as elicits multiple biological activities in a paracrine or autocrine manner. Among them the most characteristic biological activity of AM is a very powerful hypotensive activity caused by dilatation of resistance vessels. A sensitive and specific radioimmunoassay demonstrated that AM circulates in blood and occurs in a variety of tissues. Plasma AM levels elevate in various diseases including cardiovascular and renal disorders or septic shock. Thus, AM may be involved in pathophysiological processes in these diseases, especially in disorders controlling circulation and body fluid. In this short review, the history of AM and proadrenomedullin N-terminal 20 peptide (PAMP) will be reviewed with special references to biological properties and function, receptors, gene engineering and clinical viewpoints. This review includes oral presentations from the aforementioned symposium; some of which have not yet been published. These unpublished oral presentations are quoted in this paper from the abstracts of this symposium.

Preproadrenomedullin, which consists of 185 amino acids and contains a 21-amino acid signal peptide, is processed to synthesize proadrenomedullin and finally AM. In the proadrenomedullin, a unique twenty amino acid sequence followed by a typical amidation signal known as Gly-Lys-Arg, is included in the N-terminal region. This novel 20 residues peptide with carboxyl terminus of Arg-CONH2 is also present in vivo and is termed “proadrenomedullin N-terminal 20 peptide (PAMP).” PAMP elicits a potent hypotensive activity in anesthetized rats.

Although widely distributed in the adenophypophysis and the neural lobe of pituitary glands, AM and PAMP occur in cell-specific, but not overlapping, patterns in the anterior pituitary. This cell-specific expression of each peptide may be explained by differences in posttranslational processing of AM gene. As such, potential pituitary specific transcription factor binding sites, gonadotropic-specific element (GSE) and a binding site for steroidogenic factor-l (SF-1) are found in the 5flanking region of human and mouse AM gene.  SF-1 is a member of the steroid receptor superfamily that has been shown necessary for gonadotrope differentiation within the pituitary. In addition, one putative binding sequence of Pit-1 has been reported in mouse AM gene promoter position.

A specific AM binding protein (AMBP-1) in human plasma was isolated and the purified protein was identified as human complement factor H. AM and factor H interaction may interfere with the radioimmunoassay quantification of circulating AM. Factor H enhances AM-mediated induction of cAMP in fibroblast; augments the AM-mediated growth of a cancer cell line; and suppresses the bactericidal capability of AM on Escherichia coli. Conversely, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I. The augmentation of AM actions indicates that AMBP may facilitate the binding of AM to its receptor. In addition, the existence of AMBP suggests that large amounts of AM may circulate bound to this plasma protein.

In rat vascular smooth muscle cells, the CGRP, CGRP1 receptor antagonist, competitively inhibits the intracellular accumulation of cAMP induced by AM. Vasodilation of the rat mesenteric vascular bed elicited by AM and CGRP is also blocked by CGRP. Similar effects of CGRP are observed in the isolated rat heart and its microvasculature. Thus, CGRP1 receptor can mediate some effects of AM, but AM has a low affinity at CGRP2 receptor. Two distinct AM labeled bands with a molecular weight of 120 and 70 kDa was reported in the cultured rat vascular smooth muscle cell membrane. Therefore, the binding specificity and characteristics of the AM receptor may differ regionally by organ or tissue.

Two more RAMP proteins, RAMP2 and RAMP3, were discovered from database searches. These proteins share approximately 30% homology with RAMP1. Co-expression of RAMP2 or RAMP3 with CRLR appears to constitute AM receptor. RAMP2 and RAMP3 are indistinguishable in terms of AM binding. The RAMPs are required to transport CRLR to the plasma membrane. RAMP1 presents CRLR as a mature glycoprotein at the cell surface to form a CGRP receptor. However, receptors transported by RAMP2 or RAMP3 are core glycosylated and then become AM receptors. Three putative N-glycosylation sites Asn 60, Asn 112 and Asn 117 are present in the amino-terminal extracellular domain of the human CRLR. When the glycosylation of a myc-tagged CRLR was inhibited, specific 125I-CGRP and -AM binding were blocked in parallel. Substitution of the Asn 117 by threonine abolished CGRP and AM binding in the face of intact N-glycosylation and cell surface expression. RAMPs are accessory proteins of CTR and CRLR at the cell surface where they define AM, amylin, calcitonin and CGRP specificity.

The receptor component protein (RCP) was cloned on the basis of its ability to potentiate the endogenous Xenopus oocyte CGRP receptor. RCP is a cytosolic protein with no similarity to RAMPs, consists of a hydrophobic 146 amino acids and is obtained from the Corti organ of guinea pig. RCF plays an essential role for signal-transduction of CGRP and AM, and interacts with CRLR directly within the cells. Thus, a functional AM or CGRP receptor seems to consist of at least three proteins: CRLR, RAMP and RCP, coupling the receptor to the intracellular signal-transduction pathway.

By using a chimera of the CRLR and green fluorescent protein (GFP), the study demonstrated that CRLR-GFP failed to generate responses to CGRP or AM without RAMP2 or RAMP3 in HEK 293 cells. When coexpressed with RAMP2 or RAMP3, CRLR-GFP appeared on the cell membrane and activated an intracellular cAMP production and calcium mobilization. Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs.

The discovery of RAMPs has promoted our understandingthat some of the biological activities of AM are blocked by CGRP receptor antagonist, whereas other biological activities are blocked only by AM receptor antagonist, which indicates the possible existence of AM receptor in dual nature. RAMP association with CRLR traffics this receptor to the cell surface where individual RAMPs dictate the expression of unique phenotypes such as CGRP receptor or AM receptors. Apart from receptor trafficking and glycosylation, the RAMPs may interact directly with the receptors in the cell surface modifying their affinities for the ligands.

Since AM was discovered by monitoring the elevating activity of cAMP in rat platelets, cAMP appears to be its major second messenger. Dose-dependent intracellular production of cAMP induced by AM has been confirmed in various tissues and cells. Moreover, information on the role of NO in alternative signal-transduction pathways for AM is available.

The vasodilating effect of AM is reduced by the blockade of NO synthetase activity with NG-nitro-L-arginine methylester (L-NAME), indicating that NO may at least partly contribute to the AM-induced vasodilation. However, the degree of NO contribution to vasodilation varies depending upon the organ or tissue and the species. NO synthetase inhibitor in the pulmonary vascular beds of rat significantly attenuates the AM-induced vasodilation, but it does not occur in cats. Thus, NO seems to be an important AM mediator despite regional and interspecies variation.

In bovine aortic endothelial cells, AM increases intracellular ionic calcium (Ca2+) and causes the accumulation of cAMP. This increase in intracellular Ca2+ may be involved in the activation of phospholipase C, thereby producing inducible NO synthetase and subsequently NO. NO transferred to medial smooth muscle cells may activate cGMP-mediating smooth muscle cells vasodilatation. In contrast, AM lowers both cytosolic Ca2+ and Ca2+ sensitivity in smooth muscle cells of pig coronary arteries and intracellular Ca2+ in rat renal arterial smooth muscle cells.

Among the multi-functional properties of AM, the most characteristic one is an intensive, long-lasting hypotension that is dose-dependent in humans, rats, rabbits, dogs, cats and sheep. AM dilates resistance vessels in the kidneys, brain, lung, hindlimbs in animals as well as in the mesentery. Moreover, AM elicits relaxation of ring preparations of the aorta and cerebral arteries. An i.v. injection of human AM to conscious sheep causes a dose dependent fall of blood pressure, an increase in heart rate and cardiac output with a small reduction in stroke volume, as well as a marked decrease in total peripheral resistance. Coronary blood flow increases in parallel with the increase in coronary conductance. These cardiovascular responses return to the control level by 40 min after the injection.

The low-dose infusion of AM administered to conscious sheep on a low-salt diet antagonizes the vasopressor actions of administered angiotensin II while stimulating cardiac output and heart rate. AM may control cardiovascular homeostasis in part through antagonism of the vasopressor action of angiotensin II. AM inhibits the secretion of endothelin-1 from the vascular endothelial cells and proliferation of vascular smooth muscle cells. In the cultured cardiomyocytes as well as cardiac fibroblasts, AM inhibits protein synthesis in these cells in an autocrine or a paracrine manner, which may result in modulating the cardiac growth. AM inhibits bronchial constriction induced by acetylcholine or histamine in a dose-dependent  manner, indicating the important role of AM on airway function and its usefulness for the management of bronchial asthma. AM inhibits secretion of aldosterone from the adrenal cortex. When infused directly into the adrenal arterial supply of conscious sheep, AM directly inhibits the acute stimulation of aldosterone by angiotensin II,  KCl and ACTH while not affecting basal or chronic aldosterone secretion or cortisol secretion stimulated by ACTH. AM co-exists in insulin-producing cells and it inhibits insulin secretion dose-dependently in isolated rat islets.

The N-terminal region of preproadrenomedullin, the precursor of AM, contains a unique 20-residue sequence followed by Gly-Lys-Arg, a typical amidation signal, which was termed as proadrenomedullin N-terminal 20 peptide (PAMP). PAMP was purified from porcine adrenal medulla and human pheochromo-cytoma by using radioimmunoassay for the peptide and its complete amino acid sequence was determined. In addition to the original form of PAMP [1–20], PAMP [9–20] has recently been purified from the bovine adrenal medulla. The amino acid sequences of both forms of PAMP are identical to amino acid sequences deduced by cDNA analysis and their carboxyl terminus of Arg is amidated. The distribution of PAMP is similar to that of human AM, due to the fact that PAMP as well as human AM is biosynthesized from an AM precursor.

AM is processed from its precursor, proadrenomedullin, as the intermediate or immature form, AM-glycine (AM[1–52]-COOH, immature AM). Subsequently, immature AM is converted to the biologically active mature form, AM [1–52]-CONH2 (mature AM) by enzymatic amidation. The AM circulating in the human blood stream (total AM), thus, consists of both mature AM and immature AM. In earlier studies, plasma AM levels were measured by using radioimmunoassay recognizing the entire AM molecule (AM [1–52]), which reflects plasma total AM levels, as previously described.

In healthy volunteers severe exercise elevates the plasma AM levels with an increase in plasma norepinephrine and exaggerated sympathetic nerve activity. In heart transplant recipients, maximal exercise induces an increase in plasma AM that is inversely related to mean blood pressure. AM, therefore, may participate in blood pressure regulation during exercise even after heart transplantation.

When compared with healthy controls, the plasma AM levels are increased in patients with a variety of diseases: congestive heart failure, myocardial infarction, renal diseases, hypertensive diseases, diabetes mellitus, acute phase of stroke, and septic shock.

Adrenomedullin and central cardiovascular regulation

Meghan M. Taylor, Willis K. Samson
Peptides 22 (2001) 1803–1807 PII: S0196-9781(01)00522-8

Adrenomedullin gene products have been localized to neurons in brain that innervate sites known to be important in the regulation of cardiovascular function. Those sites also have been demonstrated to possess receptors for the peptide and central administrations of adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) elevate blood pressure and heart rate in both conscious and anesthetized animals. The accumulated evidence points to a role of the sympathetic nervous system in these cardiovascular effects. These sympathostimulatory actions of AM and PAMP have been hypothesized to be cardioprotective in nature and to reflect the central nervous system (CNS) equivalent of the direct cardiostimulatory effects of the peptides in the periphery. This review summarizes the most recent data on the CNS actions of the adrenomedullin gene-derived peptides and suggests future strategies for the elucidation of the physiologic relevance of the already demonstrated, pharmacologic actions of these peptides.

Adrenomedullin and related peptides: receptors and accessory proteins

Roman Muff, Walter Born, Jan A. Fischer
Peptides 22 (2001) 1765–1772  PII: S0196-9781(01)00515-0
Adrenomedullin (AM), α- and β-calcitonin gene-related peptide (CGRP), amylin and calcitonin (CT) are structurally and functionally related peptides. The structure of a receptor for CT (CTR) was elucidated in 1991 through molecular cloning, but the structures of the receptors for the other three peptides had yet to be elucidated. The discovery of receptor-activity-modifying proteins (RAMP) 1 and -2 and their co-expression with an orphan receptor, calcitonin receptor-like receptor (CRLR) has led to the elucidation of functional CGRP and AM receptors, respectively. RAMP1 and -3 which are co-expressed with CTR revealed two amylin receptor isotypes. Molecular interactions between CRLR and RAMPs are involved in their transport to the cell surface. Heterodimeric complexes between CRLR or CTR and RAMPs are required for ligand recognition.

Pharmacological profiles of receptors of the adrenomedullin peptidefamily
AMR AM>CGRP>>amylin=CT
CTR CT>amylin>>CGRP=AM
CGRPR CGRP>AM>>amylin=CT
AmylinR AmylinsCT­CGRP>>hCT>AM

Specific AM binding sites have been identified in many tissues including the heart, blood vessels, lung and spleen. Based on pharmacological evidence two receptor isotypes have been distinguished, for instance in rat astrocytes and NG108–15 cells. One AM receptor isotype recognizes CGRP and CGRP(8–37). The other receptor isotype specific for the AM ligand and antagonized by AM(22–52) does not recognize CGRP to any great extent. Both isotypes of the receptors have been shown to interact poorly with amylin and CT (Table). Biological actions of AM include vaso- and bronchodilation, and CNS transmitted inhibition of water intake.

CGRP receptors are widely distributed in the nervous and cardiovascular systems. To date, two isotypes have been described. On pharmacological evidence, CGRP1 receptors, such as those identified in human SK-N-MC neuroblastoma cells, recognize intact CGRP and CGRP(8–37) with similar potency, unlike a linear analog lacking the disulfide bridge. CGRP2 receptors,
on the other hand, interact with the linear analog but not with CGRP(8–37). These CGRP receptor isotypes cross-react with AM to some extent, but only minimally with amylin and CT. CGRP shares potent vasodilatory actions with AM, and has chronotropic and inotropic actions in the heart. The ionotropic actions are indirectly brought about via activation of the sympathetic nervous system. There is evidence to suggest the existence of α- or β-CGRP preferring receptor isotypes in both the central nervous system and peripheral tissues.

RAMP1, -2 and -3 are widely expressed, suggesting that RAMPs may have
important functions beyond those of the adrenomedullin family of receptors. To this end, RAMP1 and -3 are thought to reduce cell surface expression of angiotensin (AT) AT1 and AT2 receptors.

RAMP2 and CRLR are expressed in vascular smooth muscle cells, and RAMP1 expression was increased by dexamethasone. Moreover, increased levels of RAMP2 and CRLR were observed in the kidney and heart of rats with obstructive nephropathy and congestive heart failure, respectively. RAMP2
and CRLR levels were reduced, and RAMP3 levels were increased during lipopolysaccharide induced sepsis in rats.

The GABAB receptor 1 is retained as an immature glycoprotein in the cytosol unless co-expressed with GABAB receptor 2 isotype. Heterodimers of fully functional opioid receptors δ and κ result in a novel receptor displaying binding and functional properties distinct from those of the δ or κ receptors alone. Heterodimerization therefore facilitates receptor expression and defines ligand specificity also in G protein-coupled receptor families A and C. Moreover, heterodimers of metabotropic glutamate 1receptor (family C) and adenosine A1 receptors (family A) have been observed. As yet there is no evidence for homo or heterodimerization of family B receptors. Cysteines conserved in the extracellular N-terminal domain in all the receptors of family B and RAMPs suggest that RAMPs are truncated forms of receptors that interact as heterodimers with CRLR and CTR.

The discovery of RAMPs in combination with CRLR and CTR has led to the molecular identification of CGRP1, CGRP/amylin, AM and amylin receptor complexes. The physiological advantage of heterodimers between seven transmembrane domain receptors and the RAMPs required for the functional expression of the adrenomedullin, CGRP and amylin receptors remains to be demonstrated.

Angiotensin II, From Vasoconstrictor to Growth Factor: A Paradigm Shift

Sasa Vukelic, Kathy K. Griendling
Circ Res. 2014;114:754-757
http://dx.doi.org:/10.1161/CIRCRESAHA.114.303045

Angiotensin II (Ang II) is today considered as one of the essential factors in the pathophysiology of cardiovascular disease, producing acute hemodynamic and chronic pleiotropic effects. Although now it is widely accepted that these chronic effects are important, Ang II was initially considered only a short-acting, vasoactive hormone. This view was modified a quarter of a century ago when Dr Owens and his group published an article in Circulation Research with initial evidence that Ang II can act as a growth factor that regulates cell hypertrophy. They showed in a series of elegant experiments that Ang II promotes hypertrophy and hyperploidy of cultured rat aortic smooth muscle cells. However, Ang II had no effect on hyperplasia. These findings led to a paradigm shift in our understanding of the roles of growth factors and vasoactive substances in cardiovascular pathology and helped to redirect basic and clinical renin–angiotensin system research during the next 25 years. Ang II is now known to be a pleiotropic hormone that uses multiple signaling pathways to influence most processes that contribute to the development and progression of cardiovascular diseases, ranging from hypertrophy, endothelial dysfunction, cardiac remodeling, fibrosis, and inflammation to oxidative stress.

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Biomarker Guided Therapy

Writer and Curator: Larry H. Bernstein, MD, FCAP

Novel serum protein biomarker panel revealed by mass spectrometry and its prognostic value in breast cancer

Liping Chung, K Moore, L Phillips, FM Boyle, DJ Marsh and RC Baxter
Breast Cancer Research 2014, 16:R63
http://breast-cancer-research.com/content/16/3/R63

Introduction: Serum profiling using proteomic techniques has great potential to detect biomarkers that might improve diagnosis and predict outcome for breast cancer patients (BC). This study used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) to identify differentially expressed  proteins in sera from BC and healthy volunteers (HV), with the
goal  of developing a new prognostic biomarker panel.
Methods: Training set serum samples from 99 BC and 51 HV subjects were applied to four adsorptive chip surfaces (anion-exchange, cation-exchange, hydrophobic, and metal affinity) and analyzed by time-of-flight MS. For validation, 100 independent BC serum samples and 70 HV samples were analyzed similarly. Cluster analysis of protein spectra was performed to identify protein patterns related to BC and HV groups. Univariate and multivariate statistical analyses were used to develop a protein panel to distinguish breast cancer sera from healthy sera, and its prognostic potential was evaluated.
Results: From 51 protein peaks that were significantly up- or downregulated in BC patients by univariate analysis, binary logistic regression yielded five protein peaks that together classified BC and HV with a receiver operating characteristic (ROC) area-under-the-curve value of 0.961. Validation on an independent patient cohort confirmed the five-protein parameter (ROC value 0.939). The five-protein parameter showed positive association with large tumor size (P = 0.018) and lymph node involvement (P = 0.016). By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, immunoprecipitation and western blotting the proteins were identified as a fragment of apolipoprotein H (ApoH), ApoCI, complement C3a, transthyretin, and ApoAI. Kaplan-Meier analysis on 181 subjects after median follow-up of >5 years demonstrated that the panel significantly predicted disease-free survival (P = 0.005), its efficacy apparently greater in women with estrogen receptor (ER)-negative tumors (n = 50, P = 0.003) compared to ER-positive (n = 131, P = 0.161), although the influence of ER status needs to be confirmed after longer follow-up.
Conclusions: Protein mass profiling by MS has revealed five serum proteins which, in combination, can distinguish between serum from women with breast cancer and healthy control subjects with high sensitivity and specificity. The five-protein panel significantly predicts recurrence-free survival in women with ER-negative tumors and may have value in the management of these patients.

Variants of uncertain significance in BRCA: a harbinger of ethical and policy issues to come?

Jae Yeon Cheon, Jessica Mozersky and Robert Cook-Deegan
Genome Medicine 2014, 6:121
http://genomemedicine.com/content/6/12/121

After two decades of genetic testing and research, the BRCA1 and BRCA2 genes are two of the most well-characterized genes in the human genome. As a result, variants of uncertain significance (VUS; also called variants of unknown significance) are reported less frequently than for genes that have been less thoroughly studied. However, VUS continue to be uncovered, even for BRCA1/2. The increasing use of multi-gene panels and whole-genome and whole-exome sequencing will lead to higher rates of VUS detection because more genes are being tested, and most genomic loci have been far less intensively characterized than BRCA1/2. In this article, we draw attention to ethical and policy-related issues that will emerge. Experience garnered from BRCA1/2 testing is a useful introduction to the challenges of detecting VUS in other genetic testing contexts, while features unique to BRCA1/2 suggest key differences between the BRCA experience and the current challenges of multi-gene panels in clinical care. We propose lines of research and policy development, emphasizing the importance of pooling data into a centralized open-access database for the storage of gene variants to improve VUS interpretation. In addition, establishing ethical norms and regulated practices for sharing and curating data, analytical algorithms, interpretive frameworks and patient re-contact are important policy areas.

The Significance of Normal Pretreatment Levels of CA125 (<35 U/mL) in Epithelial Ovarian Carcinoma

Joseph Menczer,  Erez Ben-Shem,  Abraham Golan, and Tally Levy
Rambam Maimonides Med J 2015;6 (1):e0005. http://dx.doi.org:/10.5041/RMMJ.10180

Objective: To assess the association between normal CA125 levels at diagnosis of epithelial ovarian carcinoma (EOC) with prognostic factors and with outcome.
Methods: The study group consisted of histologically confirmed EOC patients with normal pretreatment CA125 levels, and the controls consisted of EOC patients with elevated (≥35 U/mL) pretreatment CA125 levels, diagnosed and treated between 1995 and 2112. Study and control group patients fulfilled the following criteria: 1) their pretreatment CA125 levels were assessed; 2) they had full standard primary treatment, i.e. cytoreductive surgery and cisplatin-based chemotherapy; and 3) they were followed every 2–4 months during the first two years and every 4–6 months thereafter.
Results: Of 114 EOC patients who fulfilled the inclusion criteria, 22 (19.3%) had normal pretreatment CA125 levels. The control group consisted of the remaining 92 patients with ≥35 U/mL serum CA125 levels pretreatment. The proportion of patients with early-stage and low-grade disease, with optimal cytoreduction, and with platin-sensitive tumors was significantly higher in the study group than in the control group. The progression-free survival (PFS) and overall survival (OS) were significantly higher in the study group than in the control group on univariate analysis but not on multivariate analysis.

Higher gene expression variability in the more aggressive subtype of chronic lymphocytic leukemia

Simone Ecker, Vera Pancaldi, Daniel Rico and Alfonso Valencia
Genome Medicine (2015) 7:8 http://dx.doi.org:/10.1186/s13073-014-0125-z

Background: Chronic lymphocytic leukemia (CLL) presents two subtypes which have drastically different clinical outcomes, IgVH mutated (M-CLL) and IgVH unmutated (U-CLL). So far, these two subtypes are not associated to clear differences in gene expression profiles. Interestingly, recent results have highlighted important roles for heterogeneity, both at the genetic and at the epigenetic level in CLL progression.
Methods: We analyzed gene expression data of two large cohorts of CLL patients and quantified expression variability across individuals to investigate differences between the two subtypes using different measures and statistical tests. Functional significance was explored by pathway enrichment and network analyses. Furthermore, we implemented a random forest approach based on expression variability to classify patients into disease subtypes.
Results: We found that U-CLL, the more aggressive type of the disease, shows significantly increased variability of gene expression across patients and that, overall, genes that show higher variability in the aggressive subtype are related to cell cycle, development and inter-cellular communication. These functions indicate a potential relation between gene expression variability and the faster progression of this CLL subtype. Finally, a classifier based on gene expression variability was able to correctly predict the disease subtype of CLL patients.
Conclusions: There are strong relations between gene expression variability and disease subtype linking significantly increased expression variability to phenotypes such as aggressiveness and resistance to therapy in CLL.

The Emerging Roles of Thyroglobulin

Yuqian Luo, Yuko Ishido, Naoki Hiroi, Norihisa Ishii, and Koichi Suzuki
Advances in Endocrinology 2014, Article ID 189194, 7 pages http://dx.doi.org/10.1155/2014/189194

Thyroglobulin (Tg), the most important and abundant protein in thyroid follicles, is well known for its essential role in thyroid hormone synthesis. In addition to its conventional role as the precursor of thyroid hormones, we have uncovered a novel function of Tg as an endogenous regulator of follicular function over the past decade. The newly discovered negative feedback effect of Tg on follicular function observed in the rat and human thyroid provides an alternative explanation for the observation of follicle heterogeneity. Given the essential role of the regulatory effects of Tg, we consider that dysregulation of normal Tg function is associated with multiple human thyroid diseases including autoimmune thyroid disease and thyroid cancer. Additionally, extrathyroid Tg may serve a regulatory function in other organs. Further exploration of Tg action, especially at the molecular level, is needed to obtain a better understanding of both the physiological and pathological roles of Tg.

The GUIDE-IT trial will help doctors find a new standard of care for heart failure.

Heart failure affects more than 25 million people worldwide, including 5.8 million in the United States and 6.9 million in Europe. About one to two percent of adults in developed countries have been diagnosed with heart failure; this increases to more than 10 percent in people over age 70. Moreover, heart failure accounts for more than 17 percent of Medicare spending and about 5 percent of total US healthcare spending. The cost to society in the US is about 30 billion dollars a year—and rising.

For people hospitalized due to heart failure, the outlook isn’t encouraging. Following discharge, one in four patients is likely to be back in the hospital in less than a month. With every acute heart failure event that requires readmission, the chances of dying from the disease increase.

Heart failure occurs when the heart is unable to fill with or pump sufficient blood to meet the needs of the body. Some heart failure symptoms—shortness of breath, fatigue and fluid buildup—which are present in other health problems. Heart failure may develop from coronary artery disease, high blood pressure, cardiomyopathy, heart valve disease, arrhythmias, viral or bacterial infections, and congenital heart defects. As a consequence, these patients often have additional diseases (comorbidities) and managing heart failure can be extremely challenging.

There have been no new drugs for heart failure in more than a decade. The last breakthrough was cardiac resynchronization therapy, a device and not a drug. The goals of therapy are to treat heart failure’s underlying causes, reduce symptoms, improve the patient’s quality of life and keep the disease from getting worse.

More than a pump

The heart isn’t just a muscle pumping blood through the body. It is also an endocrine gland that secretes peptides and hormones. When the heart is failing, its stressed cells release larger amounts of substances known as natriuretic peptides, including N-terminal prohormone brain natriuretic peptide, or NT-proBNP.

Roche’s NT-proBNP test measures the levels of this peptide and helps doctors to determine whether patients are suffering from heart failure and to assess their prognosis. Most recently, NT-proBNP has also been shown to help physicians guide and adjust the patient’s drug therapy. The objective of the pivotal GUIDE-IT trial is to demonstrate the efficacy and safety of NT-proBNP guided heart failure therapy.

Sponsored by the National Institutes of Health (NIH), the GUIDE-IT trial will help doctors answer important questions about NT-proBNP’s impact on medical care. About 1100 patients are enrolled in this robustly powered, randomized controlled trial comparing NT-proBNP guided therapy on top of standard care versus standard care alone in high-risk heart failure patients. Its primary endpoint is time to cardiovascular death or first heart failure hospitalization.

With the NT-proBNP biomarker, doctors can create personalized treatment plans for patients to substantially reduce mortality and morbidity. It can be viewed as a companion diagnostic that works with all the drugs recommended by the major guidelines.

Finding new answers

GUIDE-IT will last five years and involve approximately 45 trial sites in the United States. The first group of patients will be enrolled by the end of 2012.

“We need to take a more strategic approach if we are going to meet the AHA/ASA’s 2020 goal of reducing heart failure hospitalizations by 20 percent,” Dr. O’Connor, Chief of the Division of Cardiovascular Medicine at Duke Heart Center in Durham, North Carolina, said at a media briefing held in October at Roche Diagnostics International in Rotkreuz, Switzerland.
The relative and combined ability of: high-sensitivity cardiac troponin T, and N-terminal pro-B-type natriuretic Peptide – to predict cardiovascular events and death in patients with type 2 diabetes.

Hillis GS; Welsh P; Chalmers J; Perkovic V; Chow CK; Li Q; Jun M; Neal B; et al.
http://reference.medscape.com/medline/abstract/24089534?src=wnl_ref_prac_diab

OBJECTIVE Current methods of risk stratification in patients with type 2 diabetes are suboptimal. The current study assesses the ability of N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin T (hs-cTnT) to improve the prediction of cardiovascular events and death in patients with type 2 diabetes.
RESEARCH DESIGN AND METHODS A nested case-cohort study was performed in 3,862 patients who participated in the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial. RESULTS Seven hundred nine (18%) patients experienced a major cardiovascular event (composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke) and 706 (18%) died during a median of 5 years of follow-up. In Cox regression models, adjusting for all established risk predictors, the hazard ratio for cardiovascular events for NT-proBNP was 1.95 per 1 SD increase (95% CI 1.72, 2.20) and the hazard ratio for hs-cTnT was 1.50 per 1 SD increase (95% CI 1.36, 1.65). The hazard ratios for death were 1.97 (95% CI 1.73, 2.24) and 1.52 (95% CI 1.37, 1.67), respectively. The addition of either marker improved 5-year risk classification for cardiovascular events (net reclassification index in continuous model, 39% for NT-proBNP and 46% for hs-cTnT). Likewise, both markers greatly improved the accuracy with which the 5-year risk of death was predicted. The combination of both markers provided optimal risk discrimination.
CONCLUSIONS NT-proBNP and hs-cTnT appear to greatly improve the accuracy with which the risk of cardiovascular events or death can be estimated in patients with type 2 diabetes.

Genetics and Heart Failure: A Concise Guide for the Clinician

Cécile Skrzynia, Jonathan S. Berg, Monte S. Willis and Brian C. Jensen
Current Cardiology Reviews, 2013; 9.

Abstract: The pathogenesis of heart failure involves a complex interaction between genetic and environmental factors. Genetic factors may influence the susceptibility to the underlying etiology of heart failure, the rapidity of disease progression, or the response to pharmacologic therapy. The genetic contribution to heart failure is relatively minor in most multifactorial cases, but more direct and profound in the case of familial dilated cardiomyopathy. Early studies of genetic risk for heart failure focused on polymorphisms in genes integral to the adrenergic and renin-angiotensin-aldosterone system. Some of these variants were found to increase the risk of developing heart failure, and others appeared to affect the therapeutic response to neurohormonal antagonists. Regardless, each variant individually confers a relatively modest increase in risk and likely requires complex interaction with other variants and the environment for heart failure to develop. Dilated cardiomyopathy frequently leads to heart failure, and a genetic etiology increasingly has been recognized in cases previously considered to be “idiopathic”. Up to 50% of dilated cardiomyopathy cases without other cause likely are due to a heritable genetic mutation. Such mutations typically are found in genes encoding sarcomeric proteins and are inherited in an autosomal dominant fashion. In recent years, rapid advances in sequencing technology have improved our ability to diagnose familial dilated cardiomyopathy and those diagnostic tests are available widely. Optimal care for the expanding population of patients with heritable heart failure involves counselors and physicians with specialized training in genetics, but numerous online genetics resources are available to practicing clinicians.

Cardiac Troponin Testing Is Overused after the Rule-In or Rule-Out of Myocardial Infarction

Olaia Rodriguez Fraga, Y Sandoval, SA Love, ZJ McKinney, MAM Murakami, SW Smith, FS Apple
Clinical Chemistry 2015; 61:2 http://dx.doi.org:/10.1373/clinchem.2014.232694

No good studies have systematically evaluated appropriate clinical utilization of cardiac troponin testing in the clinical setting of the rule-in and rule-out of myocardial infarction (MI). Our collective 100-plus years of clinical and laboratory experience suggested that provider test ordering and use of cardiac troponin has been excessive after a diagnosis of MI or no MI has been determined. There is no evidence that supports continuation of cardiac troponin testing after a diagnosis is made.

Number of cTnI results demonstrating excessive orders by diagnosis

Number of cTnI results demonstrating excessive orders by diagnosis

Time and Frequency Domain Analysis of Heart Rate Variability and their orrelations in Diabetes Mellitus
T. Ahamed Seyd, V. I. Thajudin Ahamed, Jeevamma Jacob, Paul Joseph K
Intl J Biolog and Life Sciences 2008; 4(1)

Diabetes mellitus (DM) is frequently characterized by autonomic nervous dysfunction. Analysis of heart rate variability (HRV) has become a popular noninvasive tool for assessing the activities of autonomic nervous system (ANS). In this paper, changes in ANS
activity are quantified by means of frequency and time domain analysis of R-R interval variability. Electrocardiograms (ECG) of 16 patients suffering from DM and of 16 healthy volunteers were recorded. Frequency domain analysis of extracted normal to normal interval (NN interval) data indicates significant difference in very low frequency (VLF) power, low frequency (LF) power and high frequency (HF) power, between the DM patients and control group. Time domain measures, standard deviation of NN interval (SDNN), root mean square of successive NN interval differences (RMSSD), successive NN intervals differing more than 50 ms (NN50 Count), percentage value of NN50 count (pNN50), HRV triangular index and triangular interpolation of NN intervals (TINN) also show significant difference between the DM patients and control group.

Power Spectral Density of the RR interval of a 55 year old healthy volunteer

Power Spectral Density of the RR interval of a 55 year old healthy volunteer

Power Spectral Density of the RR interval of a 55 year old healthy volunteer

Power Spectral Density of the RR interval of a 62 year old woman suffering

Power Spectral Density of the RR interval of a 62 year old woman suffering

Power Spectral Density of the RR interval of a 62 year old woman suffering
from diabetes for the last 15 years

HRV analysis has gained much importance in recent years, as a technique employed to explore the activity of ANS, and as an important early marker for identifying different pathological conditions. DM is a disease in which the cardiac autonomic activity is progressively compromised. Our investigation indicates that different time domain and frequency domain measures of HRV would be able to provide valuable information regarding the autonomic dysfunction to DM.

Time domain and frequency domain analysis of the RR interval variability of diabetic and normal subjects shows that there is significant difference in these measures for DM patients with respect to normal subjects. Variation of the HRV parameters indicates changes in ANS activity of DM patients. This can provide valid information regarding autonomic neuropathy in people with diabetes. It may be noted that these methods can detect changes before clinical signs appear. So we can expect that these measures enable early detection and treatment/subsequent management of patients and thus can avoid acute and chronic complications.

Multiparametric diagnostics of cardiomyopathies by microRNA signatures

Christine S. Siegismund & Maria Rohde & Uwe Kühl & Dirk Lassner
Microchim Acta 2014   http://dx.doi.org:/10.1007/s00604-014-1249-y

The diagnosis of cardiomyopathies by endomyocardial biopsy analysis is the gold standard for confirmation of causative reasons but is failing if a sample does not contain the area of interest due to focal pathology. Biopsies are revealing an extract of the current situation of the heart muscle only, and the need for global organ-specific or systemic markers is obvious in order to minimize sampling errors. Global markers like specific gene expression signatures in myocardial tissue may therefore reflect the focal situation or condition of the whole myocardium. Besides gene expression profiles, microRNAs (miRNAs) represent a new group of stable biomarkers that are detectable both in tissue and body fluids. Such miRNAs may serve as cardiological biomarkers to characterize inflammatory processes, to confirm viral infections, and to differentiate various forms of infection.
The predictive power of single miRNAs for diagnosis of complex diseases may be further increased if several distinctly deregulated candidates are combined to form a specific miRNA signature. Diagnostic systems that generate disease related miRNA profiles are based on microarrays, bead-based oligo sorbent assays, or on assays based on real-time polymerase chain reactions and placed on microfluidic cards or nanowell plates. Multiparametric diagnostic systems that can measure differentially expressed miRNAs may become the diagnostic tool of the future due to their predictive value with respect to clinical course, therapeutic decisions, and therapy monitoring. We discuss here specific merits, limitations and the potential of currently available analytical platforms for diagnostics of heart muscle diseases based on miRNA profiling.

Predictive value of plasma galectin-3 levels in heart failure with reduced and preserved ejection fraction

Rudolf A. de Boer, DJA Lok, T Jaarsma, P van der Meer, AA Voors, et al.
Annals Med, 2011; 43: 60–68 http://dx.doi.org:/10.3109/07853890.2010.538080

We studied the prognostic value of base-line galectin-3 in a large HF cohort, with preserved and reduced left ventricular ejection fraction (LVEF), and compared this to other biomarkers.
Methods. We studied 592 HF patients who had been hospitalized for HF and were followed for 18 months. The primary end-point was a composite of all-cause mortality and HF hospitalization.
Results. A doubling of galectin-3 levels was associated with a hazard ratio (HR) of 1.97 (1.62–2.42) for the primary outcome (P= 0.001). After correction for age, gender, BNP, eGFR, and diabetes the HR was 1.38 (1.07–1.78; P= 0.015). Galectin-3 levels were correlated with higher IL -6 and CRP levels (P= 0.002). Changes of galectin-3 levels after 6 months did not add prognostic information to the base-line value (n= 291); however, combining plasma galectin-3 and BNP levels increased prognostic value over either biomarker alone (ROC analysis, P = 0.05). The predictive value of galectin-3 was stronger in patients with preserved LVEF (n= 114) compared to patients with reduced LVEF (P= 0.001).
Conclusions. Galectin-3 is an independent marker for outcome in HF and appears to be particularly useful in HF patients with preserved LVEF.

Criteria for the use of omics-based predictors in clinical trials

Lisa M. McShane, MM Cavenagh, TG Lively, DA Eberhard, et al.
Nature  17 Oct 2013; 502: 317-320. http://dx.doi.org:/10.1038/nature12564

The US National Cancer Institute (NCI), in collaboration with scientists representing multiple areas of expertise relevant to ‘omics’-based test development, has developed a checklist of criteria that can be used to determine the readiness of omics-based tests for guiding patient care in clinical trials. The checklist criteria cover issues relating to specimens, assays, mathematical modelling, clinical trial design, and ethical, legal and regulatory aspects. Funding bodies and journals are encouraged to consider the checklist, which they may find useful for assessing study quality and evidence strength. The checklist will be used to evaluate proposals for NCI-sponsored clinical
trials in which omics tests will be used to guide therapy.

M-Atrial Natriuretic Peptide and Nitroglycerin in a Canine Model of Experimental Acute Hypertensive Heart Failure: Differential Actions of 2 cGMP Activating Therapeutics.

Paul M McKie, Alessandro Cataliotti, Tomoko Ichiki, S Jeson Sangaralingham, Horng H Chen, John C Burnett
J Am Heart Assoc 01/2014; 3(1):e000206. http://dx.doi.org:/10.1161/JAHA.113.000206

Systemic hypertension is a common characteristic in acute heart failure (HF). This increasingly recognized phenotype is commonly associated with renal dysfunction and there is an unmet need for renal enhancing therapies. In a canine model of HF and acute vasoconstrictive hypertension we characterized and compared the cardiorenal actions of M-atrial natriuretic peptide (M-ANP), a novel particulate guanylyl cyclase (pGC) activator, and nitroglycerin, a soluble guanylyl cyclase (sGC) activator.
HF was induced by rapid RV pacing (180 beats per minute) for 10 days. On day 11, hypertension was induced by continuous angiotensin II infusion. We characterized the cardiorenal and humoral actions prior to, during, and following intravenous M-ANP (n=7), nitroglycerin (n=7), and vehicle (n=7) infusion. Mean arterial pressure (MAP) was reduced by M-ANP (139±4 to 118±3 mm Hg, P<0.05) and nitroglycerin (137±3 to 116±4 mm Hg, P<0.05); similar findings were recorded for pulmonary wedge pressure (PCWP) with M-ANP (12±2 to 6±2 mm Hg, P<0.05) and nitroglycerin (12±1 to 6±1 mm Hg, P<0.05). M-ANP enhanced renal function with significant increases (P<0.05) in glomerular filtration rate (38±4 to 53±5 mL/min), renal blood flow (132±18 to 236±23 mL/min), and natriuresis (11±4 to 689±37 mEq/min) and also inhibited aldosterone activation (32±3 to 23±2 ng/dL, P<0.05), whereas nitroglycerin had no significant (P>0.05) effects on these renal parameters or aldosterone activation.
Our results advance the differential cardiorenal actions of pGC (M-ANP) and sGC (nitroglycerin) mediated cGMP activation. These distinct renal and aldosterone modulating actions make M-ANP an attractive therapeutic for HF with concomitant hypertension, where renal protection is a key therapeutic goal.

Genome-Wide Association Study of a Heart Failure Related Metabolomic Profile Among African Americans in the Atherosclerosis Risk in Communities (ARIC) Study

Bing Yu, Y Zheng, D Alexander, TA Manolio, A Alonso, JA Nettleton, & E Boerwinkle
Genet Epidemiol 2013; 00:1–6, http://dx.doi.org:/10.1002/gepi.21752

Both the prevalence and incidence of heart failure (HF) are increasing, especially among African Americans, but no large-scale, genome-wide association study (GWAS) of HF-related metabolites has been reported. We sought to identify novel genetic variants that are associated with metabolites previously reported to relate to HF incidence. GWASs of three metabolites identified previously as risk factors for incident HF (pyroglutamine, dihydroxy docosatrienoic acid, and X-11787, being either hydroxy-leucine or hydroxy-isoleucine) were performed in 1,260 African Americans free of HF at the baseline examination of the Atherosclerosis Risk in Communities (ARIC) study. A significant association on chromosome 5q33 (rs10463316, MAF = 0.358, P-value = 1.92 × 10−10) was identified for pyroglutamine. One region on chromosome 2p13 contained a nonsynonymous substitution in N-acetyltransferase 8 (NAT8) was associated with X-11787 (rs13538, MAF = 0.481, P-value = 1.71 × 10−23). The smallest P-value for dihydroxy docosatrienoic acid was rs4006531 on chromosome 8q24 (MAF = 0.400, P-value = 6.98 × 10−7). None of the above SNPs were individually associated with incident HF, but a genetic risk score (GRS) created by summing the most significant risk alleles from each metabolite detected 11% greater risk of HF per allele. In summary, we identified three loci associated with previously reported HF-related metabolites. Further use of metabolomics technology will facilitate replication of these findings in independent samples.

Global Left Atrial Strain Correlates with CHADS2 Risk Score in Patients with Atrial Fibrillation

SK Saha, PL Anderson, G Caracciolo, A Kiotsekoglou, S Wilansky, S Govind, et al.
J Am Soc Echocardiogr 2011; 24(5): 506-512.
http://dx.doi.org:/10.1016/j.echo.2011.02.012

Background: The aim of this cross-sectional study was to explore the association between echocardiographic parameters and CHADS2 score in patients with nonvalvular atrial fibrillation (AF).
Methods: Seventy-seven subjects (36 patients with AF, 41 control subjects) underwent standard twodimensional, Doppler, and speckle-tracking echocardiography to compute regional and global left atrial (LA) strain.
Results: Global longitudinal LA strain was reduced in patients with AF compared with controls (P < .001) and was a predictor of high risk for thromboembolism (CHADS2 score $ 2; odds ratio, 0.86; P = .02). LA strain indexes showed good interobserver and intraobserver variability. In sequential Cox models, the prediction of hospitalization and/or death was improved by addition of global LA strain and indexed LA volume to CHADS2 score (P = .003).
Conclusions: LA strain is a reproducible marker of dynamic LA function and a predictor of stroke risk and cardiovascular outcomes in patients with AF.

Gene Expression and Genetic Variation in Human Atria

Honghuang Lin, EV Dolmatova, MP Morley, KL Lunetta, et al.
Heart Rhythm, HRTHM5533. PII: S1547-5271(13)01226-5
http://dx.doi.org/10.1016/j.hrthm.2013.10.051

Background— The human left and right atria have different susceptibilities to develop atrial fibrillation (AF). However, the molecular events related to structural and functional changes that enhance AF susceptibility are still poorly understood.
Objective— To characterize gene expression and genetic variation in human atria.
Methods— We studied the gene expression profiles and genetic variations in 53 left atrial and 52 right atrial tissue samples collected from the Myocardial Applied Genomics Network (MAGNet) repository. The tissues were collected from heart failure patients undergoing transplantation and from unused organ donor hearts with normal ventricular function. Gene expression was profiled using the Affymetrix GeneChip Human Genome U133A Array. Genetic variation was profiled using the Affymetrix Genome-Wide Human SNP Array 6.0.
Results— We found that 109 genes were differentially expressed between left and right atrial tissues. A total of 187 and 259 significant cis-associations between transcript levels and genetic variants were identified in left and right atrial tissues, respectively. We also found that a SNP at a known AF locus, rs3740293, was associated with the expression of MYOZ1 in both left and right atrial tissues. Conclusion— We found a distinct transcriptional profile between the right and left atrium, and extensive cis-associations between atrial transcripts and common genetic variants. Our results implicate MYOZ1 as the causative gene at the chromosome 10q22 locus for AF.

Atrial Natriuretic Peptide Single Nucleotide Polymorphisms in Patients with Nonfamilial Structural Atrial Fibrillation

Pietro Francia, A Ricotta, A Frattari, R Stanzione, A Modestino, et al.
Clinical Medicine Insights: Cardiology 2013:7 153–159
http://dx.doi.org:/10.4137/CMC.S12239

Background: Atrial natriuretic peptide (ANP) has antihypertrophic and antifibrotic properties that are relevant to AF substrates. The −G664C and rs5065 ANP single nucleotide polymorphisms (SNP) have been described in association with clinical phenotypes, including hypertension and left ventricular hypertrophy. A recent study assessed the association of early AF and rs5065 SNPs in low-risk subjects. In a Caucasian population with moderate-to-high cardiovascular risk profile and structural AF, we conducted a case-control study to assess whether the ANP −G664C and rs5065 SNP associate with nonfamilial structural AF.
Methods: 168 patients with nonfamilial structural AF and 168 age- and sex-matched controls were recruited. The rs5065 and −G664C ANP SNPs were genotyped.
Results: The study population had a moderate-to-high cardiovascular risk profile with 86% having hypertension, 23% diabetes, 26% previous myocardial infarction, and 23% left ventricular systolic dysfunction. Patients with AF had greater left atrial diameter (44 ± 7
vs. 39 ± 5 mm; P , 0.001) and higher plasma NTproANP levels (6240 ± 5317 vs. 3649 ± 2946 pmol/mL; P , 0.01). Odds ratios (ORs)
for rs5065 and −G664C gene variants were 1.1 (95% confidence interval [CI], 0.7–1.8; P = 0.71) and 1.2 (95% CI, 0.3–3.2; P = 0.79), respectively, indicating no association with AF. There were no differences in baseline clinical characteristics among carriers and noncarriers of the −664C and rs5065 minor allele variants.
Conclusions: We report lack of association between the rs5065 and −G664C ANP gene SNPs and AF in a Caucasian population of patients with structural AF. Further studies will clarify whether these or other ANP gene variants affect the risk of different subphenotypes of AF driven by distinct pathophysiological mechanisms.

N-terminal proBNP and mortality in hospitalized patients with heart failure and preserved vs. reduced systolic function: data from the prospective Copenhagen Hospital Heart Failure Study (CHHF)

Kirk, M. Bay, J. Parnerc, K. Krogsgaard, T.M. Herzog, S. Boesgaard, et al.
Eur Journal Heart Failure 6 (2004) 335–341
http://dx.doi.org:/10.1016/j.ejheart.2004.01.002

Preserved systolic function among heart failure patients is a common finding, a fact that has only recently been fully appreciated. The aim of the present study was to examine the value of NT-proBNP to predict mortality in relation to established risk factors among consecutively hospitalised heart failure patients and secondly to characterise patients in relation to preserved and reduced systolic function. Material: At the time of admission 2230 consecutively hospitalised patients had their cardiac status evaluated through determinations of NT-proBNP, echocardiography, clinical examination and medical history. Follow-up was performed 1 year later in all patients. Results: 161 patients fulfilled strict diagnostic criteria for heart failure (HF). In this subgroup of patients 1-year mortality was approximately 30% and significantly higher as compared to the remaining non-heart failure population (approx. 16%). Using univariate analysis left ventricular ejection fraction (LVEF), New York Heart Association classification (NYHA) and plasma levels of NT-proBNP all predicted mortality independently. However, regardless of systolic function, age and NYHA class, risk-stratification was provided by measurements of NT-proBNP. Having measured plasma levels of NT-proBNP, LVEF did not provide any additional prognostic information on mortality among heart failure patients (multivariate analysis).
Conclusion: The results show that independent of LVEF, measurements of NT-proBNP add additional prognostic information. It is concluded that NT-proBNP is a strong predictor of 1-year mortality in consecutively hospitalised patients with heart failure with preserved as well as reduced systolic function.

N-terminal pro-B-type natriuretic peptide and the prediction of primary cardiovascular events: results from 15-year follow-up of WOSCOPS

Paul Welsh, Orla Doolin, Peter Willeit, Chris Packard, Peter Macfarlane, et al.
Eur Heart Journal 2014. http://eurheartj.oxfordjournals.org/

Aims: To test whether N-terminal pro-B-type natriuretic peptide (NT-proBNP) was independently associated with, and improved the prediction of, cardiovascular disease (CVD) in a primary prevention cohort.
Methods and results:  In the West of Scotland Coronary Prevention Study (WOSCOPS), a cohort of middle-aged men with hypercholesterolemia at a moderate risk of CVD, we related the baseline NT-proBNP (geometric mean 28 pg/mL) in 4801 men to the risk of CVD over 15 years during which 1690 experienced CVD events. Taking into account the competing risk of non-CVD death, NT-proBNP was associated with an increased risk of all CVD [HR: 1.17 (95% CI: 1.11–1.23) per standard deviation increase in log NT-proBNP] after adjustment for classical and clinical cardiovascular risk factors plus C-reactive protein. N-terminal pro-B-type natriuretic peptide was more strongly related to the risk of fatal [HR: 1.34 (95% CI: 1.19–1.52)] than non-fatal CVD [HR: 1.17 (95% CI: 1.10–1.24)] (P ¼ 0.022). The addition of NT-proBNP to traditional risk factors improved the C-index (+0.013; P , 0.001). The continuous net reclassification index improved with the addition of NT-proBNP by 19.8% (95% CI: 13.6–25.9%) compared with 9.8% (95% CI: 4.2–15.6%) with the addition of C-reactive protein. N-terminal pro-B-type natriuretic peptide correctly reclassified 14.7% of events, whereas C-reactive protein correctly reclassified 3.4% of events. Results were similar in the 4128 men without evidence of angina, nitrate prescription, minor ECG abnormalities, or prior cerebrovascular disease.
Conclusion: N-terminal pro-B-type natriuretic peptide predicts CVD events in men without clinical evidence of CHD, angina, or history of stroke, and appears related more strongly to the risk for fatal events. N-terminal pro-B-type natriuretic peptide also provides moderate risk discrimination, in excess of that provided by the measurement of C-reactive protein.

Effect of B-type natriuretic peptide-guided treatment of chronic heart failure on total mortality and hospitalization: an individual patient meta-analysis

Richard W. Troughton, Christopher M. Frampton, Hans-Peter Brunner-La Rocca,
Matthias Pfisterer, Luc W.M. Eurlings, Hans Erntell, Hans Persson, et al.
Eur Heart J 2014; 35: 1559–1567 http://dx.doi.org:/10.1093/eurheartj/ehu090

Aims Natriuretic peptide-guided (NP-guided) treatment of heart failure has been tested against standard clinically guided care in multiple studies, but findings have been limited by study size. We sought to perform an individual patient data metaanalysis to evaluate the effect of NP-guided treatment of heart failure on all-cause mortality.
Methods and results
Eligible randomized clinical trials were identified from searches of Medline andEMBASEdatabases and the Cochrane Clinical
Trials Register. The primary pre-specified outcome, all-cause mortality was tested using a Cox proportional hazards regression model that included study of origin, age (< 75 or ≥75 years), and left ventricular ejection fraction (LVEF, ≤45 or .45%) as covariates. Secondary endpoints included heart failure or cardiovascular hospitalization. Of 11 eligible studies, 9 provided individual patient data and 2 aggregate data. For the primary endpoint individual data from 2000 patients were included, 994 randomized to clinically guided care and 1006 to NP-guided care. All-cause mortality was significantly reduced by NP-guided treatment [hazard ratio = 0.62 (0.45–0.86);
P = 0.004] with no heterogeneity between studies or interaction with LVEF. The survival benefit from NP-guided therapy was seen in younger ( <75 years) patients [0.62 (0.45–0.85); P = 0.004] but not older (≥75 years) patients [0.98 (0.75–1.27); P = 0.96]. Hospitalization due to heart failure [0.80 (0.67–0.94); P = 0.009] or cardiovascular disease [0.82 (0.67–0.99); P = 0.048]was significantly lower in NP-guided patients with no heterogeneity between studies and no interaction with age or LVEF.
Conclusion: Natriuretic peptide-guided treatment of heart failure reduces all-cause mortality in patients aged < 75 years and overall reduces heart failure and cardiovascular hospitalization.

Diagnostic and prognostic evaluation of left ventricular systolic heart failure by plasma N-terminal pro-brain natriuretic peptide concentrations in a large sample of the general population

B A Groenning, I Raymond, P R Hildebrandt, J C Nilsson, M Baumann, F Pedersen
Heart 2004;90:297–303. http://dx.doi.org:/10.1136/hrt.2003.026021

Objective: To evaluate N-terminal pro-brain natriuretic peptide (NT-proBNP) as a diagnostic and prognostic marker for systolic heart failure in the general population.
Design: Study participants, randomly selected to be representative of the background population, filled in a heart failure questionnaire and underwent pulse and blood pressure measurements, electrocardiography, echocardiography, and blood sampling and were followed up for a median (range) period of 805 (6021171) days.
Setting: Participants were recruited from four randomly selected general practitioners and were examined in a Copenhagen university hospital.
Patients: 382 women and 290 men in four age groups (50259 (n = 174); 60269 (n = 204); 70279 (n = 174); > 80 years (n = 120)).
Main outcome measures: Value of NT-proBNP in evaluating patients with symptoms of heart failure and impaired left ventricular (LV) systolic function; prognostic value of NT-proBNP for mortality and hospital admissions.
Results: In 38 (5.6%) participants LV ejection fraction (LVEF) was (40%. NT-proBNP identified patients with symptoms of heart failure and LVEF (40% with a sensitivity of 0.92, a specificity of 0.86, positive and negative predictive values of 0.11 and 1.00, and area under the curve of 0.94. NT-proBNP was the strongest independent predictor of mortality (hazard ratio (HR) = 5.70, p = 0.0001), hospital admissions for heart failure (HR = 13.83, p = 0.0001), and other cardiac admissions (HR = 3.69, p = 0.0001). Mortality (26 v 6, p = 0.0003), heart failure admissions (18 v 2, p = 0.0002), and admissions for other cardiac causes (44 v 13, p = 0.0001) were significantly higher in patients with NTproBNP above the study median (32.5 pmol/l). Conclusions: Measurement of NT-proBNP may be useful as a screening tool for systolic heart failure in the general population.

Copeptin—Marker of Acute Myocardial Infarction

Martin Möckel & Julia Searle
Curr Atheroscler Rep 2014; 16:421 http://dx.doi.org:/10.1007/s11883-014-0421-5

The concentration of copeptin, the C-terminal part of pro-arginine vasopressin, has been shown to increase early after acute and severe events. Owing to complementary pathophysiology and kinetics, the unspecific marker copeptin, in combination with highly cardio-specific troponin, has been evaluated as an early-rule-out strategy for acute myocardial infarction in patients presenting with signs and symptoms of acute coronary syndrome. Overall, most studies have reported a negative predictive value between 97 and 100 % for the diagnosis of acute myocardial infarction in low- to intermediate-risk patients with suspected acute coronary syndrome. Additionally, a recent multicenter, randomized process study, where patients who tested negative for copeptin and troponin were discharged from the emergency department, showed that the safety of the new process was comparable to that of the current standard process. Further interventional trials and data from registries are needed to ensure the effectiveness and patient benefit of the new strategy.

The role of copeptin as a diagnostic and prognostic biomarker for risk stratification in the emergency department

Christian H Nickel1, Roland Bingisser and Nils G Morgenthaler
BMC Medicine 2012, 10:7 http://www.biomedcentral.com/1741-7015/10/7

The hypothalamic-pituitary-adrenal axis is activated in response to stress. One of the activated hypothalamic hormones is arginine vasopressin, a hormone involved in hemodynamics and osmoregulation. Copeptin, the C-terminal part of the arginine vasopressin precursor peptide, is a sensitive and stable surrogate marker for arginine vasopressin release. Measurement of copeptin levels has been shown to be useful in a variety of clinical scenarios, particularly as a prognostic marker in patients with acute diseases such as lower respiratory tract infection, heart disease and stroke. The measurement of copeptin levels may provide crucial information for risk stratification in a variety of clinical situations. As such, the emergency department appears to be the ideal setting for its potential use. This review summarizes the recent progress towards determining the prognostic and diagnostic value of copeptin in the emergency department.

Variability of the Transferrin Receptor 2 Gene in AMD

Daniel Wysokinski, Janusz Blasiak, Mariola Dorecka, Marta Kowalska, et al.
Disease Markers 2014, Article ID 507356, 8 pages http://dx.doi.org/10.1155/2014/507356

Oxidative stress is a major factor in the pathogenesis of age-related macular degeneration (AMD). Iron may catalyze the Fenton reaction resulting in overproduction of reactive oxygen species. Transferrin receptor 2 plays a critical role in iron homeostasis and variability in its gene may influence oxidative stress and AMD occurrence. To verify this hypothesis we assessed the association between  polymorphisms of the TFR2 gene and AMD. A total of 493AMDpatients and 171matched controls were genotyped for the two polymorphisms of the TFR2 gene: c.1892C>T (rs2075674) and c.−258+123T>C (rs4434553). We also assessed the modulation of some AMD risk factors by these polymorphisms.The CC and TT genotypes of the c.1892C>T were associated with AMD occurrence but the latter only in obese patients. The other polymorphism was not associated with AMD occurrence, but the CC genotype was correlated with an increasing AMD frequency in subjects with BMI < 26. The TT genotype and the T allele of this polymorphism decreased AMD occurrence in subjects above 72 years, whereas the TC genotype and the C allele increased occurrence of AMD in this group.The c.1892C>T and c.−258+123T>C polymorphisms of the TRF2 gene may be associated with AMD occurrence, either directly or by modulation of risk factors.

Urinary N-Acetyl-beta-D-glucosaminidase as an Early Marker for Acute Kidney Injury in Full-Term Newborns with Neonatal Hyperbilirubinemia

Bangning Cheng, Y Jin, G Liu, Z Chen, H Dai, and M Liu
Disease Markers 2014, Article ID 315843, 6 pages http://dx.doi.org/10.1155/2014/315843

Purpose. To investigate renal function estimated by markers in full-term newborns with hyperbilirubinemia.
Methods. A total of 332 full-term newborns with hyperbilirubinemia and 60 healthy full-term newborns were enrolled. Total serum bilirubin, serum creatinine (Cr), serum blood urea nitrogen (BUN), serum cystatin C (Cys-C), urinary beta-2-microglobulin (𝛽2MG) index, and urinary N-acetyl-beta-D-glucosaminidase (NAG) index were measured before and after treatment. All newborns were divided into three groups according to total serum bilirubin levels: group 1 (221-256), group 2 (256-342), and group 3 (>342). Results. The control group and group 1 did not differ significantly in regard to serum Cr, serum BUN, serum Cys-C, urinary 𝛽2MG index, and urinary NAG index. Urinary NAG index in group 2 was significantly higher than that in control group (𝑃 < 0.001). Between control group and group 3, serum Cys-C, urinary 𝛽2MG index, and urinary NAG index differed significantly. The significant positive correlation between total serum bilirubin and urinary NAG index was found in newborns when total serum bilirubin level was more than 272 𝜇mol/L.
Conclusions. High unconjugated bilirubin could result in acute kidney injury in full-term newborns. Urinary NAG might be the suitable marker for predicting acute kidney injury in full-term newborns with hyperbilirubinemia.

Urinary C-peptide creatinine ratio detects absolute insulin deficiency in Type 2 diabetes.

S V Hope, A G Jones, E Goodchild, M Shepherd, R E J Besser, B Shields, T McDonald, B A Knight, A Hattersley

Department of Geriatrics, Royal Devon and Exeter NHS Foundation Trust; NIHR Exeter Clinical Research Facility, University of Exeter.

Diabetic Medicine (impact factor: 2.9). 05/2013; http://dx.doi.org:/10.1111/dme.12222

Source: PubMed

ABSTRACT AIMS: To determine the prevalence and clinical characteristics of absolute insulin deficiency in long-standing Type 2 diabetes, using a strategy based on home urinary C-peptide creatinine ratio measurement.
METHODS: We assessed the urinary C-peptide creatinine ratios, from urine samples taken at home 2 h after the largest meal of the day, in 191 insulin-treated subjects with Type 2 diabetes (diagnosis age ≥45 years, no insulin in the first year). If the initial urinary C-peptide creatinine ratio was ≤0.2 nmol/mmol (representing absolute insulin deficiency), the assessment was repeated. A standardized mixed-meal tolerance test with 90-min stimulated serum C-peptide measurement was performed in nine subjects with a urinary C-peptide creatinine ratio ≤ 0.2 nmol/mmol (and in nine controls with a urinary C-peptide creatinine ratio >0.2 nmol/mmol) to confirm absolute insulin deficiency.
RESULTS: A total of 2.7% of participants had absolute insulin deficiency confirmed by a mixed-meal tolerance test. They were identified initially using urinary C-peptide creatinine ratio: 11/191 subjects (5.8%) had two consistent urinary C-peptide creatinine ratios ≤ 0.2 nmol/mmol; 9/11 subjects completed a mixed-meal tolerance test and had a median stimulated serum C-peptide of 0.18nmol/l. Five out of nine subjects had stimulated serum C-peptide <0.2 nmol/l and 9/9 subjects with urinary C-peptide creatinine ratio >0.2 had endogenous insulin secretion confirmed by the mixed-meal tolerance test. Compared with subjects with a urinary C-peptide creatinine ratio >0.2 nmol/mmol, those with confirmed absolute insulin deficiency had a shorter time to insulin treatment (median 2.5 vs. 6 years, P=0.005) and lower BMI (25.1 vs. 29.1kg/m(2) , P=0.04). Two out of five patients were glutamic acid decarboxylase autoantibody-positive.
CONCLUSIONS: Absolute insulin deficiency may occur in long-standing Type 2 diabetes, and cannot be reliably predicted by clinical features or autoantibodies. Its recognition should help guide treatment, education and management. The urinary C-peptide creatinine ratio is a practical non-invasive method to aid detection of absolute insulin deficiency, with a urinary C-peptide creatinine ratio > 0.2 nmol/mmol being a reliable indicator of retained endogenous insulin secretion.

Unlocking Biomarkers’ Full Potential

David Daniels, Ph.D.     genengnews  Feb 1, 2013 (Vol. 33, No. 3)

http://www.genengnews.com/gen-articles/unlocking-biomarkers-full-potential/4700/

Biomarker research and development has evolved over the past years from looking for a single marker (e.g., PSA) linked to a disease state to looking for a panel of markers that can capture the heterogeneity inherent in both the disease and the impacted patient population.

That is one of the key messages to be delivered at GTC’s “Biomarkers Summit” next month. Across the board, resources are being focused on the delivery of more precise, quantifiable biomarkers with predictive value in therapeutic decisions and for the prognosis of illness.

“Our focus on biomarker development is the recognition that the new products need to provide cost savings for the already strapped healthcare systems rather than just be cost effective,” shares Paul Billings, M.D., Ph.D., CMO at Life Technologies.

“We have built a new medical sciences group to address the needs of the multiple delivery systems in the world—from the sophisticated medical clinics in the developed world to the nurse-run shanty clinic in the third world. Providing tools for equitable access to quality diagnosis, on assay platforms that can provide care for all patients, is our goal.”

Life Tech’s medical sciences division has been built by acquisition of Pinpoint Genomics, Navigenics, and Compendia, and collaborations with partners such as Ingenuity Systems and CollabRx. The division is focused on taking the tools that have been used in the life science laboratories and providing molecular diagnostic data to the clinic. The intent is to deliver data in a valuable format that can be used by the molecular pathologist or the treating physician.

The division is developing the Pervenio™ Lung RS assay, a 14-gene expression profile that serves as a risk stratifier that uses a weighted algorithm for the expressed biomarkers within the tumor biopsy, a first-of-its-kind prognostic test for lung cancer, the firm reports.

Initially, tests will be offered as a service through Life Tech’s CLIA laboratory. Then, from the performance lessons learned, Life Tech’s will develop a simpler assay platform, with FDA approval, that can be dispersed globally without reduction of the essential content in the biomarker panel. The focus is on the workflow—screening for known mutations using established easy-to-use assay platforms, like RT-PCR. Should the screen not produce useful results, clinicians can search for new mutations via discovery platforms like next-gen sequencing (NGS).

http://www.genengnews.com/Media/images/Article/thumb_Sequenom_LungCartaPanel1722631391.jpg

Sequenom’s LungCarta panel of 214 somatic mutations in 26 tumor suppressors and oncogenes covers highly mutated pathways in lung adenocarcinomas.

At Sequenom, the company provides both the tools (DNA mass spectrometry and reagents) for confirmatory biomarker development as well as serving on the front lines as a diagnostic service provider (CLIA lab). The beauty of DNA mass spec is that it can process multiplexed PCR samples (10–60 loci) in a method that is quantitative when used for profiling tumor biopsies that are either archival or fresh tissue.

Given a tumor sample with multiple somatic mutations, the instrument enables the determination of the homogeneity of the cells, in which case the mutations will have the same allele frequency. Accuracy, as measured by coefficient of variance, is less than 2%. Despite this level of sensitivity, the mass spec can only be used as a confirmatory tool looking for known mutations. Discovery is best done using DNA sequencing. DNA mass spec can also be used to study methylation in tumor samples.

“In the not-too-distant future, we will be looking for mutations in plasma samples rather than biopsies,” predicts Charles Cantor, Ph.D., CSO at Sequenom.

“The key is to look noninvasively for mutations within plasma samples such that we can potentially catch the disease state earlier, rather than after tumor formation. Regardless of the tumor type, this approach will enable us to monitor therapeutic response and metastatic potential noninvasively. DNA mass spec is an ultrasensitive detection product that can detect somatic mutations at levels of 1 per 1,000. This level of sensitivity is critical for the future of plasma screening. NGS technology is not that sensitive.”

Sequenom’s CLIA lab is using automated DNA mass spec to provide three different test protocols: (1) carrier screening for cystic fibrosis looking at more than 100 different mutations, (2) adult macular degeneration progression using an SNP test with 13 loci, and (3) a noninvasive test for Rh compatibility between a mother and her unborn fetus.

http://www.genengnews.com/Media/images/Article/thumb_Illumina_HiSeq_Scientist2141841107.jpg

Scientists are using Illumina’s HiSeq system to discover molecular biomarkers that may provide opportunities for early detection of a range of diseases.

Sequenom has also set up an NGS facility within a CLIA lab in San Diego using Illumina’s HiSEQ platform. The NGS platform has been set up for noninvasive aneuploidy detection of maternal plasma (10 cc sample) looking at chromosomes 13, 18, and 21. The lab says it has analyzed more than 40,000 samples this year and is planning to increase that volume up to 100,000 samples per year. Most of these samples come from the U.S., but given the development of a new blood collection tube that allows for 72-hour ambient shipping, the lab is looking to increase the number of samples from outside the U.S.

Drug Development

During drug development, biomarkers function as pharmacodynamic markers to help assess the mechanism of action of a drug candidate, to define the downstream biological pathway, and to determine whether the drug is engaging the target with the anticipated biological effect. Later, biomarkers help determine whether a drug is effective using the tested regime (route of delivery, dosage level, and length of exposure time).

Following early development, the second stage is to use biomarkers to help segment patients for clinical trials. Part of the consideration here is how heterogeneous the disease is; are there homogeneous subsets of patients that will respond differentially to the drug based on different mechanisms of the disease?

“Biomarker research is focused on on- target effects,” says Nick Dracopoli, Ph.D., vp, head of oncology biomarkers at Janssen Research and Development, a J&J company.

“We look at indications and at patients with those indications that are most likely to respond to the drug candidates we’re developing. For oncology biomarkers, germ-line effects are weaker indicators than somatic changes in the tumor. As a consequence, SNP-based, genome-wide association studies are not very useful. It is better to focus on molecular changes within the tumor and define gene expression profiles and epigenetic modifications that correlate with the tumor phenotype. We are increasingly tracking patient immune response, particularly as more immuno-oncology products are moving into the drug development pipeline.”

The number of biomarkers being developed varies from project to project. But it is very clear that to be successful in the clinic, the biomarkers and the assays need to be of low complexity. Of the 10 to 12 companion diagnostics that have been approved by the FDA to date, all measure the status of the drug target (on-target markers). For example, EGFR measures the level of receptor expression; Braf and Kras markers measure the presence of the mutation and translocation in the ALK gene measures gene knockout.

It is important to realize that molecular profiles for first-in-class drugs are not optimal because they are based on only a few patients. Consequently they have weak predictive value overall.

“Aside from that rule of thumb, if you have a greater than 50% response rate for your drug, it is unlikely that you need a biomarker to predict response. Biomarker utility is best for drugs that would have a difficult road to approval, where it is critical to enrich for the subpopulation of responders. For example, Pfizer’s crizontinib was approved for non-small-cell lung patients but is only effective for 5% of all patients. If Pfizer was unable to demonstrate the relationship between activation of the ALK gene and disease, this inhibitor would not have been approved,” says Dr. Dracopoli.

“Drugs that are more broadly active can come to market without a companion diagnostic test. There is always a balance between the predictive values of the biomarker test and the response rates to treatment. That is, we should not treat if the chance of response is only 3–5%, rather than if it were 50% where the patient would want to take the chance if the drug were safe.”

An important take-home message is that mutations are not unique to an indication. So if you find a driver mutation in indications for which the drug has not been approved, you could discover new indications for the drug.

“At the end of the day, this is what cancer is—heterogeneous,” says Dr. Dracopoli. “We’d all love to treat one cancer with one drug and at one dose, but the story is more complex. The future of oncology is around understanding the molecular heterogeneity or underlying molecular pathology of the disease and the diversity of it, and then treat each patient accordingly.”

Clinical Considerations

“Given the complexity of biology,” says Achim Plum, Ph.D., principal consultant, Siemens, “whether is it cancer, metabolic disease, or any other disease state, we have been forced to move away from the idea that a single biomarker can capture the entire ‘story’ or mechanistic view of any disease. Hence newly developed biomarkers will be made up of a panel of markers that serve as a profile. In addition, with the sheer volume of DNA and protein analytics data, the clinic will need to employ software tools and algorithms to help the decision making.”

The task of getting broad profiling technologies that are analytical into a clinical setting and making them routine is difficult but not insurmontable. This will take a collaborative effort, something that Siemens among others are looking to develop. The key is to avoid technology hype and to establish good reliable software to process the data for decision making. “Data is not knowledge, and knowledge is not automatically decision making.”

As an academic, Daniel Chan, Ph.D., has a view of the whole value chain for biomarkers from discovery to development to use in the clinic. Dr. Chan holds the titles of professor in pathology, oncology, radiology, and urology, and is the director of the clinical chemistry division lab at Johns Hopkins Hospital.

Given his perspective from discovery to clinical use, Dr. Chan indicated that from the clinical point of view, “we need more markers.” He oversees the discovery of new biomarkers in his research lab, their validation in his translational research lab, and finally their utility in practice in his clinical chemistry lab. He is a strong advocate for collaboration of biomarker development from discovery to verification and validation to incorporation within the clinical practice.

Beyond the use of biomarkers for patient stratification and correlation between marker and therapeutic choice, as is the focus of the biopharma industry, for the clinic the use of biomarkers is for prevention and early detection. The earlier the detection, the better the outcome. That is, provide the “cure” before you need to initiate treatment.

To be successful in the future of biomarkers, we need to look beyond the biopharma focus and expand the horizon for early detection and monitor therapy later, says Dr. Chan. He describes a roadmap of developing bridges (to bridge the knowledge gaps), gates (decision gates for go/no go decisions as to whether a development path is viable), and partnerships (to collaborate with different points of view) for efficient new biomarker development.

According to Dr. Chan, we must define the intended use of the biomarker, which identifies the specific application and sets up the clinical study and study population to meet the clinical needs. We need to define specific assays to monitor biomarkers that will work within a clinical setting, not a research lab setting that uses disease models (tissue culture cells or small animals) and not real patient samples.

“The days when single markers are sufficient (PSA for prostate cancer or troponin for cardiovascular disease) are behind us. We need to develop a panel of markers or a profile pattern to address patient population heterogeneity and disease complexity that will guide our decision-making process,” remarks Dr. Chan. “Molecular biomarkers are giving way to protein biomarkers,” he adds.

Prevention and early detection will require the use of whole-body scans, so the sampling technology and analytical tools to be developed are critical to realize this goal. Assay ease of use, automation, and analytical performance that is suitable for the clinical lab are fundamental.

“An important future goal for biomarkers,” says Dr. Billings, “is to sample circulating tumor cells or circulating DNA in blood or plasma samples as a noninvasive measure of patient status. A decline in tumor biomarkers during chemotherapy, for example, could reflect the efficacy of the therapy. In contrast, an increase in tumor biomarkers, in a patient who had previously undergone surgery and therapy, might indicate disease recurrence, and is likely to do so before a tumor mass is detectable by imaging methods.”

STAT4 Gene Polymorphisms Are Associated with Susceptibility and ANA Status in Primary Biliary Cirrhosis

Satoru Joshita, T Umemura, M Nakamura, Y Katsuyama, S Shibata, et al.
Disease Markers  2014, Article ID 727393, 8 pages http://dx.doi.org/10.1155/2014/727393

Recent genome-wide association studies suggest that genetic factors contribute to primary biliary cirrhosis (PBC) susceptibility. Although several reports have demonstrated that the interleukin (IL) 12 signaling pathway is involved in PBC pathogenesis, its precise genetic factors have not been fully clarified. Here, we performed an association analysis between IL12A, IL12RB, and signal transducer and activator of transcription 4 (STAT4) genetic variations and susceptibility to PBC. Single nucleotide polymorphisms (SNPs) were genotyped in 395 PBC patients and 458 healthy subjects of Japanese ethnicity and evaluated for associations with PBC susceptibility, anti-nuclear antibody (ANA) status, and anti-mitochondrial antibody (AMA) status. We detected significant associations with PBC susceptibility for several STAT4 SNPs (rs10168266; p = 9.4 × 10−3, rs11889341; p = 1.2 × 10−3, rs7574865; p = 4.0 × 10−4, rs8179673; p = 2.0 × 10−4, and rs10181656; p = 4.2 × 10−5). Three risk alleles (rs7574865; p = 0.040, rs8179673; p = 0.032, and rs10181656; p = 0.031) were associated with ANA status, but not with AMA positivity. Our findings confirm that STAT4 is involved in PBC susceptibility and may play a role in ANA status in the Japanese population.

Serum Omentin-1 as a Disease Activity Marker for Crohn’s Disease

Yan Lu, Li Zhou, L Liu, Yan Feng, Li Lu, X Ren, X Dong, & W Sang
Disease Markers  2014, Article ID 162517, 5 pages   http://dx.doi.org/10.1155/2014/162517

Background and Aim. It remains challenging to determine the inflammatory activity in Crohn’s disease (CD) for lack of specific laboratory markers. Recent studies suggest that serum omentin-1 is associated with inflammatory response. We aimed to assess the potential of serum omentin-1 as a marker of disease activity in CD patients.
Methods. Serum omentin-1 concentrations were determined by enzyme-linked immunosorbent assay (ELISA) in patients with CD (n = 240), functional gastrointestinal disorders (FGDs, n = 120), and healthy controls (HC, n = 60) and evaluated for correlation with disease activity. Expression of omentin-1 in colonic tissues from patients with CD was also analyzed by real-time PCR and Western blotting. Serum omentin-1 levels as an activity index were evaluated using a receiver operating characteristic (ROC) curve.
Results. Serum omentin-1 concentrations were significantly decreased in active CD patients compared with patients in remission, FGDs, and HC (all p < 0.001). Expression of omentin-1 was decreased at mRNA and protein levels in inflamed colonic tissues in active CD than that in noninflamed colonic tissues. Serum omentin-1 levels were negatively correlated with disease activity in CD, better than C-reactive protein (CRP).
Conclusion. Our results indicate that serum and colonic omentin-1 expressions are decreased in active CD patients. The correlation of serum omentin-1 with disease activity in CD is superior to that of CRP. Serum omentin-1 is a potential marker for CD disease activity.
Serum Levels of Resistin, Adiponectin, and Apelin in Gastroesophageal Cancer Patients

Dorota Diakowska, K Markocka-Mdczka, P Szelachowski, and K Grabowski
Disease Markers 2014, Article ID 619649, 8 pages   http://dx.doi.org/10.1155/2014/619649

The aim of the study was the investigation of relationship between cachexia syndrome and serum resistin, adiponectin, and apelin in patients with gastroesophageal cancer (GEC).
Material and Methods. Adipocytokines concentrations were measured in sera of 85 GEC patients and 60 healthy controls. They were also evaluated in tumor tissue and appropriate normal mucosa of 38 operated cancer patients.
Results. Resistin and apelin concentrations were significantly higher in GEC patients than in the controls. The highest resistin levels were found in cachectic patients and in patients with distant metastasis. Serum adiponectin significantly decreased in GEC patients with regional and distant metastasis. Serum apelin was significantly higher in cachectic patients than in the controls. Apelin was positively correlated with hsCRP level. Resistin and apelin levels increased significantly in tumor tissues. Weak positive correlations between adipocytokines levels in serum and in tumor tissue were observed.
Conclusions. Resistin is associated with cachexia and metastasis processes of GEC. Reduction of serum adiponectin reflects adipose tissue wasting in relation to GEC progression. Correlation of apelin with hsCRP can reflect a presumable role of apelin in systemic inflammatory response in esophageal and gastric cancer.

Serum Level of HER-2 Extracellular Domain in Iranian Patients with Breast Cancer: A Follow-up Study

Mehrnoosh Doroudchi, Abdolrasoul Talei, Helmout Modjtahedi, et al.
IJI 2005; 2(4): 191-200

Background: A soluble form of HER-2/neu extracellular domain (sHER-2) is reported to be released in the sera of metastatic breast cancer patients.
Objective: To measure the level of sHER-2 in sera of 115 breast cancer patients. Methods: Serial samples of 27 patients with metastasis, 18 non-metastatic patients, 15 patients in stage 0/I and 14 patients with accompanying benign breast disease were also included in this study.
Results: No significant difference was observed between sHER-2 level in the pre-operative sera of breast cancer patients and that of healthy individuals. Only 8 out of 27 patients whom later developed metastasis showed elevated levels of sHER-2 in their first serum sample. However, a trend of increase in the level of sHER-2 was observed in 14 (51.8%) of 27 metastatic sera before clinical diagnosis of the metastasis. A significant association between sHER-2 positive status and vascular invasion of the tumor was observed (P = 0.02). In addition, significant correlation of sHER-2 level with CEA (highest r = 0.74) and CA 15.3 (highest r = 0.74) tumor marker levels in the serial sera were observed. The mean time from sHER-2 positivity to tumor metastasis was calculated to be 98 days (range = 29-174).  Conclusion: Our results indicate that a relatively high percentage of Iranian patients with breast cancer show an elevated level of sHER-2 in their sera before clinical diagnosis of the tumor metastasis. Therefore, measuring the level of this oncoprotein, not only helps physicians in monitoring the patients during HERCEPTIN therapy, but also can be helpful in choosing more aggressive treatments at the early satges of tumor metastasis.
B-type natriuretic peptide is a biomarker for pulmonary hypertension in preterm infants with bronchopulmonary dysplasia

Alain Cuna, Jegen Kandasamy, Naomi Fineberg, Brian Sims
Research and Reports in Neonatology 2013:3 33–36
http://dx.doi.org/10.2147/RRN.S42236

Background: B-type natriuretic peptide (BNP) is a cardiac biomarker useful in screening for pulmonary hypertension (PH) in adults. It is possible that BNP may also be useful in detecting PH among preterm infants with bronchopulmonary dysplasia (BPD).
Objective: To determine the utility of BNP for identification of PH among preterm infants with BPD.
Methods: We retrospectively identified preterm infants with BPD who underwent screening echocardiography for suspected PH and had serum BNP levels measured within 10 days before or after echocardiography. Eligible infants were classified based on echocardiographic diagnosis of either PH or no PH. Median and interquartile ranges (IQR) of BNP values were compared, and area under the curve (AUC) of receiver operator characteristic (ROC) analysis was used to determine the optimum threshold value for detection of PH.
Results: Twenty-five preterm infants with BPD (mean gestational age 26.5 ± 1.7 weeks, mean birth weight 747 ± 248 g) were identified. The median difference in days between echocardiography and BNP measurement was 1 day (IQR 0–3, range 0–10 days). Based on echocardiography, 16 were diagnosed with PH and nine without PH. No significant difference in terms of gestational age, birth weight, sex, race, or respiratory support was found between the two groups. Median (IQR) BNP values of those with PH were higher than those without PH (413 [212–1178] pg/mL versus 55 [21–84] pg/mL, P , 0.001). AUC of ROC analysis showed that a BNP value of 117 pg/mL had 93.8% sensitivity and 100% specificity for detecting PH.
Conclusion: BNP estimation may be useful for screening of PH in infants with BPD.

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Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer


Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer

Author and Curator: Larry H. Bernstein, MD, FCAP

This summary is the last of a series on the impact of transcriptomics, proteomics, and metabolomics on disease investigation, and the sorting and integration of genomic signatures and metabolic signatures to explain phenotypic relationships in variability and individuality of response to disease expression and how this leads to  pharmaceutical discovery and personalized medicine.  We have unquestionably better tools at our disposal than has ever existed in the history of mankind, and an enormous knowledge-base that has to be accessed.  I shall conclude here these discussions with the powerful contribution to and current knowledge pertaining to biochemistry, metabolism, protein-interactions, signaling, and the application of the -OMICS to diseases and drug discovery at this time.

The Ever-Transcendent Cell

Deriving physiologic first principles By John S. Torday | The Scientist Nov 1, 2014
http://www.the-scientist.com/?articles.view/articleNo/41282/title/The-Ever-Transcendent-Cell/

Both the developmental and phylogenetic histories of an organism describe the evolution of physiology—the complex of metabolic pathways that govern the function of an organism as a whole. The necessity of establishing and maintaining homeostatic mechanisms began at the cellular level, with the very first cells, and homeostasis provides the underlying selection pressure fueling evolution.

While the events leading to the formation of the first functioning cell are debatable, a critical one was certainly the formation of simple lipid-enclosed vesicles, which provided a protected space for the evolution of metabolic pathways. Protocells evolved from a common ancestor that experienced environmental stresses early in the history of cellular development, such as acidic ocean conditions and low atmospheric oxygen levels, which shaped the evolution of metabolism.

The reduction of evolution to cell biology may answer the perennially unresolved question of why organisms return to their unicellular origins during the life cycle.

As primitive protocells evolved to form prokaryotes and, much later, eukaryotes, changes to the cell membrane occurred that were critical to the maintenance of chemiosmosis, the generation of bioenergy through the partitioning of ions. The incorporation of cholesterol into the plasma membrane surrounding primitive eukaryotic cells marked the beginning of their differentiation from prokaryotes. Cholesterol imparted more fluidity to eukaryotic cell membranes, enhancing functionality by increasing motility and endocytosis. Membrane deformability also allowed for increased gas exchange.

Acidification of the oceans by atmospheric carbon dioxide generated high intracellular calcium ion concentrations in primitive aquatic eukaryotes, which had to be lowered to prevent toxic effects, namely the aggregation of nucleotides, proteins, and lipids. The early cells achieved this by the evolution of calcium channels composed of cholesterol embedded within the cell’s plasma membrane, and of internal membranes, such as that of the endoplasmic reticulum, peroxisomes, and other cytoplasmic organelles, which hosted intracellular chemiosmosis and helped regulate calcium.

As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.

Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.  ….

As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.

Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.

Given that the unicellular toolkit is complete with all the traits necessary for forming multicellular organisms (Science, 301:361-63, 2003), it is distinctly possible that metazoans are merely permutations of the unicellular body plan. That scenario would clarify a lot of puzzling biology: molecular commonalities between the skin, lung, gut, and brain that affect physiology and pathophysiology exist because the cell membranes of unicellular organisms perform the equivalents of these tissue functions, and the existence of pleiotropy—one gene affecting many phenotypes—may be a consequence of the common unicellular source for all complex biologic traits.  …

The cell-molecular homeostatic model for evolution and stability addresses how the external environment generates homeostasis developmentally at the cellular level. It also determines homeostatic set points in adaptation to the environment through specific effectors, such as growth factors and their receptors, second messengers, inflammatory mediators, crossover mutations, and gene duplications. This is a highly mechanistic, heritable, plastic process that lends itself to understanding evolution at the cellular, tissue, organ, system, and population levels, mediated by physiologically linked mechanisms throughout, without having to invoke random, chance mechanisms to bridge different scales of evolutionary change. In other words, it is an integrated mechanism that can often be traced all the way back to its unicellular origins.

The switch from swim bladder to lung as vertebrates moved from water to land is proof of principle that stress-induced evolution in metazoans can be understood from changes at the cellular level.

http://www.the-scientist.com/Nov2014/TE_21.jpg

A MECHANISTIC BASIS FOR LUNG DEVELOPMENT: Stress from periodic atmospheric hypoxia (1) during vertebrate adaptation to land enhances positive selection of the stretch-regulated parathyroid hormone-related protein (PTHrP) in the pituitary and adrenal glands. In the pituitary (2), PTHrP signaling upregulates the release of adrenocorticotropic hormone (ACTH) (3), which stimulates the release of glucocorticoids (GC) by the adrenal gland (4). In the adrenal gland, PTHrP signaling also stimulates glucocorticoid production of adrenaline (5), which in turn affects the secretion of lung surfactant, the distension of alveoli, and the perfusion of alveolar capillaries (6). PTHrP signaling integrates the inflation and deflation of the alveoli with surfactant production and capillary perfusion.  THE SCIENTIST STAFF

From a cell-cell signaling perspective, two critical duplications in genes coding for cell-surface receptors occurred during this period of water-to-land transition—in the stretch-regulated parathyroid hormone-related protein (PTHrP) receptor gene and the β adrenergic (βA) receptor gene. These gene duplications can be disassembled by following their effects on vertebrate physiology backwards over phylogeny. PTHrP signaling is necessary for traits specifically relevant to land adaptation: calcification of bone, skin barrier formation, and the inflation and distention of lung alveoli. Microvascular shear stress in PTHrP-expressing organs such as bone, skin, kidney, and lung would have favored duplication of the PTHrP receptor, since sheer stress generates radical oxygen species (ROS) known to have this effect and PTHrP is a potent vasodilator, acting as an epistatic balancing selection for this constraint.

Positive selection for PTHrP signaling also evolved in the pituitary and adrenal cortex (see figure on this page), stimulating the secretion of ACTH and corticoids, respectively, in response to the stress of land adaptation. This cascade amplified adrenaline production by the adrenal medulla, since corticoids passing through it enzymatically stimulate adrenaline synthesis. Positive selection for this functional trait may have resulted from hypoxic stress that arose during global episodes of atmospheric hypoxia over geologic time. Since hypoxia is the most potent physiologic stressor, such transient oxygen deficiencies would have been acutely alleviated by increasing adrenaline levels, which would have stimulated alveolar surfactant production, increasing gas exchange by facilitating the distension of the alveoli. Over time, increased alveolar distension would have generated more alveoli by stimulating PTHrP secretion, impelling evolution of the alveolar bed of the lung.

This scenario similarly explains βA receptor gene duplication, since increased density of the βA receptor within the alveolar walls was necessary for relieving another constraint during the evolution of the lung in adaptation to land: the bottleneck created by the existence of a common mechanism for blood pressure control in both the lung alveoli and the systemic blood pressure. The pulmonary vasculature was constrained by its ability to withstand the swings in pressure caused by the systemic perfusion necessary to sustain all the other vital organs. PTHrP is a potent vasodilator, subserving the blood pressure constraint, but eventually the βA receptors evolved to coordinate blood pressure in both the lung and the periphery.

Gut Microbiome Heritability

Analyzing data from a large twin study, researchers have homed in on how host genetics can shape the gut microbiome.
By Tracy Vence | The Scientist Nov 6, 2014

Previous research suggested host genetic variation can influence microbial phenotype, but an analysis of data from a large twin study published in Cell today (November 6) solidifies the connection between human genotype and the composition of the gut microbiome. Studying more than 1,000 fecal samples from 416 monozygotic and dizygotic twin pairs, Cornell University’s Ruth Ley and her colleagues have homed in on one bacterial taxon, the family Christensenellaceae, as the most highly heritable group of microbes in the human gut. The researchers also found that Christensenellaceae—which was first described just two years ago—is central to a network of co-occurring heritable microbes that is associated with lean body mass index (BMI).  …

Of particular interest was the family Christensenellaceae, which was the most heritable taxon among those identified in the team’s analysis of fecal samples obtained from the TwinsUK study population.

While microbiologists had previously detected 16S rRNA sequences belonging to Christensenellaceae in the human microbiome, the family wasn’t named until 2012. “People hadn’t looked into it, partly because it didn’t have a name . . . it sort of flew under the radar,” said Ley.

Ley and her colleagues discovered that Christensenellaceae appears to be the hub in a network of co-occurring heritable taxa, which—among TwinsUK participants—was associated with low BMI. The researchers also found that Christensenellaceae had been found at greater abundance in low-BMI twins in older studies.

To interrogate the effects of Christensenellaceae on host metabolic phenotype, the Ley’s team introduced lean and obese human fecal samples into germ-free mice. They found animals that received lean fecal samples containing more Christensenellaceae showed reduced weight gain compared with their counterparts. And treatment of mice that had obesity-associated microbiomes with one member of the Christensenellaceae family, Christensenella minuta, led to reduced weight gain.   …

Ley and her colleagues are now focusing on the host alleles underlying the heritability of the gut microbiome. “We’re running a genome-wide association analysis to try to find genes—particular variants of genes—that might associate with higher levels of these highly heritable microbiota.  . . . Hopefully that will point us to possible reasons they’re heritable,” she said. “The genes will guide us toward understanding how these relationships are maintained between host genotype and microbiome composition.”

J.K. Goodrich et al., “Human genetics shape the gut microbiome,” Cell,  http://dx.doi.org:/10.1016/j.cell.2014.09.053, 2014.

Light-Operated Drugs

Scientists create a photosensitive pharmaceutical to target a glutamate receptor.
By Ruth Williams | The Scentist Nov 1, 2014
http://www.the-scientist.com/?articles.view/articleNo/41279/title/Light-Operated-Drugs/

light operated drugs MO1

light operated drugs MO1

http://www.the-scientist.com/Nov2014/MO1.jpg

The desire for temporal and spatial control of medications to minimize side effects and maximize benefits has inspired the development of light-controllable drugs, or optopharmacology. Early versions of such drugs have manipulated ion channels or protein-protein interactions, “but never, to my knowledge, G protein–coupled receptors [GPCRs], which are one of the most important pharmacological targets,” says Pau Gorostiza of the Institute for Bioengineering of Catalonia, in Barcelona.

Gorostiza has taken the first step toward filling that gap, creating a photosensitive inhibitor of the metabotropic glutamate 5 (mGlu5) receptor—a GPCR expressed in neurons and implicated in a number of neurological and psychiatric disorders. The new mGlu5 inhibitor—called alloswitch-1—is based on a known mGlu receptor inhibitor, but the simple addition of a light-responsive appendage, as had been done for other photosensitive drugs, wasn’t an option. The binding site on mGlu5 is “extremely tight,” explains Gorostiza, and would not accommodate a differently shaped molecule. Instead, alloswitch-1 has an intrinsic light-responsive element.

In a human cell line, the drug was active under dim light conditions, switched off by exposure to violet light, and switched back on by green light. When Gorostiza’s team administered alloswitch-1 to tadpoles, switching between violet and green light made the animals stop and start swimming, respectively.

The fact that alloswitch-1 is constitutively active and switched off by light is not ideal, says Gorostiza. “If you are thinking of therapy, then in principle you would prefer the opposite,” an “on” switch. Indeed, tweaks are required before alloswitch-1 could be a useful drug or research tool, says Stefan Herlitze, who studies ion channels at Ruhr-Universität Bochum in Germany. But, he adds, “as a proof of principle it is great.” (Nat Chem Biol, http://dx.doi.org:/10.1038/nchembio.1612, 2014)

Enhanced Enhancers

The recent discovery of super-enhancers may offer new drug targets for a range of diseases.
By Eric Olson | The Scientist Nov 1, 2014
http://www.the-scientist.com/?articles.view/articleNo/41281/title/Enhanced-Enhancers/

To understand disease processes, scientists often focus on unraveling how gene expression in disease-associated cells is altered. Increases or decreases in transcription—as dictated by a regulatory stretch of DNA called an enhancer, which serves as a binding site for transcription factors and associated proteins—can produce an aberrant composition of proteins, metabolites, and signaling molecules that drives pathologic states. Identifying the root causes of these changes may lead to new therapeutic approaches for many different diseases.

Although few therapies for human diseases aim to alter gene expression, the outstanding examples—including antiestrogens for hormone-positive breast cancer, antiandrogens for prostate cancer, and PPAR-γ agonists for type 2 diabetes—demonstrate the benefits that can be achieved through targeting gene-control mechanisms.  Now, thanks to recent papers from laboratories at MIT, Harvard, and the National Institutes of Health, researchers have a new, much bigger transcriptional target: large DNA regions known as super-enhancers or stretch-enhancers. Already, work on super-enhancers is providing insights into how gene-expression programs are established and maintained, and how they may go awry in disease.  Such research promises to open new avenues for discovering medicines for diseases where novel approaches are sorely needed.

Super-enhancers cover stretches of DNA that are 10- to 100-fold longer and about 10-fold less abundant in the genome than typical enhancer regions (Cell, 153:307-19, 2013). They also appear to bind a large percentage of the transcriptional machinery compared to typical enhancers, allowing them to better establish and enforce cell-type specific transcriptional programs (Cell, 153:320-34, 2013).

Super-enhancers are closely associated with genes that dictate cell identity, including those for cell-type–specific master regulatory transcription factors. This observation led to the intriguing hypothesis that cells with a pathologic identity, such as cancer cells, have an altered gene expression program driven by the loss, gain, or altered function of super-enhancers.

Sure enough, by mapping the genome-wide location of super-enhancers in several cancer cell lines and from patients’ tumor cells, we and others have demonstrated that genes located near super-enhancers are involved in processes that underlie tumorigenesis, such as cell proliferation, signaling, and apoptosis.

Super-enhancers cover stretches of DNA that are 10- to 100-fold longer and about 10-fold less abundant in the genome than typical enhancer regions.

Genome-wide association studies (GWAS) have found that disease- and trait-associated genetic variants often occur in greater numbers in super-enhancers (compared to typical enhancers) in cell types involved in the disease or trait of interest (Cell, 155:934-47, 2013). For example, an enrichment of fasting glucose–associated single nucleotide polymorphisms (SNPs) was found in the stretch-enhancers of pancreatic islet cells (PNAS, 110:17921-26, 2013). Given that some 90 percent of reported disease-associated SNPs are located in noncoding regions, super-enhancer maps may be extremely valuable in assigning functional significance to GWAS variants and identifying target pathways.

Because only 1 to 2 percent of active genes are physically linked to a super-enhancer, mapping the locations of super-enhancers can be used to pinpoint the small number of genes that may drive the biology of that cell. Differential super-enhancer maps that compare normal cells to diseased cells can be used to unravel the gene-control circuitry and identify new molecular targets, in much the same way that somatic mutations in tumor cells can point to oncogenic drivers in cancer. This approach is especially attractive in diseases for which an incomplete understanding of the pathogenic mechanisms has been a barrier to discovering effective new therapies.

Another therapeutic approach could be to disrupt the formation or function of super-enhancers by interfering with their associated protein components. This strategy could make it possible to downregulate multiple disease-associated genes through a single molecular intervention. A group of Boston-area researchers recently published support for this concept when they described inhibited expression of cancer-specific genes, leading to a decrease in cancer cell growth, by using a small molecule inhibitor to knock down a super-enhancer component called BRD4 (Cancer Cell, 24:777-90, 2013).  More recently, another group showed that expression of the RUNX1 transcription factor, involved in a form of T-cell leukemia, can be diminished by treating cells with an inhibitor of a transcriptional kinase that is present at the RUNX1 super-enhancer (Nature, 511:616-20, 2014).

Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization 
Andrea Sánchez-Vallet, et al.   eLife 2013;2:e00790 http://elifesciences.org/content/2/e00790#sthash.LnqVMJ9p.dpuf

LysM effector

LysM effector

http://img.scoop.it/ZniCRKQSvJOG18fHbb4p0Tl72eJkfbmt4t8yenImKBVvK0kTmF0xjctABnaLJIm9

While host immune receptors

  • detect pathogen-associated molecular patterns to activate immunity,
  • pathogens attempt to deregulate host immunity through secreted effectors.

Fungi employ LysM effectors to prevent

  • recognition of cell wall-derived chitin by host immune receptors

Structural analysis of the LysM effector Ecp6 of

  • the fungal tomato pathogen Cladosporium fulvum reveals
  • a novel mechanism for chitin binding,
  • mediated by intrachain LysM dimerization,

leading to a chitin-binding groove that is deeply buried in the effector protein.

This composite binding site involves

  • two of the three LysMs of Ecp6 and
  • mediates chitin binding with ultra-high (pM) affinity.

The remaining singular LysM domain of Ecp6 binds chitin with

  • low micromolar affinity but can nevertheless still perturb chitin-triggered immunity.

Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex.

Mutated Genes in Schizophrenia Map to Brain Networks
From www.nih.gov –  Sep 3, 2013

Previous studies have shown that many people with schizophrenia have de novo, or new, genetic mutations. These misspellings in a gene’s DNA sequence

  • occur spontaneously and so aren’t shared by their close relatives.

Dr. Mary-Claire King of the University of Washington in Seattle and colleagues set out to

  • identify spontaneous genetic mutations in people with schizophrenia and
  • to assess where and when in the brain these misspelled genes are turned on, or expressed.

The study was funded in part by NIH’s National Institute of Mental Health (NIMH). The results were published in the August 1, 2013, issue of Cell.

The researchers sequenced the exomes (protein-coding DNA regions) of 399 people—105 with schizophrenia plus their unaffected parents and siblings. Gene variations
that were found in a person with schizophrenia but not in either parent were considered spontaneous.

The likelihood of having a spontaneous mutation was associated with

  • the age of the father in both affected and unaffected siblings.

Significantly more mutations were found in people

  • whose fathers were 33-45 years at the time of conception compared to 19-28 years.

Among people with schizophrenia, the scientists identified

  • 54 genes with spontaneous mutations
  • predicted to cause damage to the function of the protein they encode.

The researchers used newly available database resources that show

  • where in the brain and when during development genes are expressed.

The genes form an interconnected expression network with many more connections than

  • that of the genes with spontaneous damaging mutations in unaffected siblings.

The spontaneously mutated genes in people with schizophrenia

  • were expressed in the prefrontal cortex, a region in the front of the brain.

The genes are known to be involved in important pathways in brain development. Fifty of these genes were active

  • mainly during the period of fetal development.

“Processes critical for the brain’s development can be revealed by the mutations that disrupt them,” King says. “Mutations can lead to loss of integrity of a whole pathway,
not just of a single gene.”

These findings support the concept that schizophrenia may result, in part, from

  • disruptions in development in the prefrontal cortex during fetal development.

James E. Darnell’s “Reflections”

A brief history of the discovery of RNA and its role in transcription — peppered with career advice
By Joseph P. Tiano

James Darnell begins his Journal of Biological Chemistry “Reflections” article by saying, “graduate students these days

  • have to swim in a sea virtually turgid with the daily avalanche of new information and
  • may be momentarily too overwhelmed to listen to the aging.

I firmly believe how we learned what we know can provide useful guidance for how and what a newcomer will learn.” Considering his remarkable discoveries in

  • RNA processing and eukaryotic transcriptional regulation

spanning 60 years of research, Darnell’s advice should be cherished. In his second year at medical school at Washington University School of Medicine in St. Louis, while
studying streptococcal disease in Robert J. Glaser’s laboratory, Darnell realized he “loved doing the experiments” and had his first “career advancement event.”
He and technician Barbara Pesch discovered that in vivo penicillin treatment killed streptococci only in the exponential growth phase and not in the stationary phase. These
results were published in the Journal of Clinical Investigation and earned Darnell an interview with Harry Eagle at the National Institutes of Health.

Darnell arrived at the NIH in 1956, shortly after Eagle  shifted his research interest to developing his minimal essential cell culture medium, still used. Eagle, then studying cell metabolism, suggested that Darnell take up a side project on poliovirus replication in mammalian cells in collaboration with Robert I. DeMars. DeMars’ Ph.D.
adviser was also James  Watson’s mentor, so Darnell met Watson, who invited him to give a talk at Harvard University, which led to an assistant professor position
at the MIT under Salvador Luria. A take-home message is to embrace side projects, because you never know where they may lead: this project helped to shape
his career.

Darnell arrived in Boston in 1961. Following the discovery of DNA’s structure in 1953, the world of molecular biology was turning to RNA in an effort to understand how
proteins are made. Darnell’s background in virology (it was discovered in 1960 that viruses used RNA to replicate) was ideal for the aim of his first independent lab:
exploring mRNA in animal cells grown in culture. While at MIT, he developed a new technique for purifying RNA along with making other observations

  • suggesting that nonribosomal cytoplasmic RNA may be involved in protein synthesis.

When Darnell moved to Albert Einstein College of Medicine for full professorship in 1964,  it was hypothesized that heterogenous nuclear RNA was a precursor to mRNA.
At Einstein, Darnell discovered RNA processing of pre-tRNAs and demonstrated for the first time

  • that a specific nuclear RNA could represent a possible specific mRNA precursor.

In 1967 Darnell took a position at Columbia University, and it was there that he discovered (simultaneously with two other labs) that

  • mRNA contained a polyadenosine tail.

The three groups all published their results together in the Proceedings of the National Academy of Sciences in 1971. Shortly afterward, Darnell made his final career move
four short miles down the street to Rockefeller University in 1974.

Over the next 35-plus years at Rockefeller, Darnell never strayed from his original research question: How do mammalian cells make and control the making of different
mRNAs? His work was instrumental in the collaborative discovery of

  • splicing in the late 1970s and
  • in identifying and cloning many transcriptional activators.

Perhaps his greatest contribution during this time, with the help of Ernest Knight, was

  • the discovery and cloning of the signal transducers and activators of transcription (STAT) proteins.

And with George Stark, Andy Wilks and John Krowlewski, he described

  • cytokine signaling via the JAK-STAT pathway.

Darnell closes his “Reflections” with perhaps his best advice: Do not get too wrapped up in your own work, because “we are all needed and we are all in this together.”

Darnell Reflections - James_Darnell

Darnell Reflections – James_Darnell

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Recent findings on presenilins and signal peptide peptidase

By Dinu-Valantin Bălănescu

γ-secretase and SPP

γ-secretase and SPP

Fig. 1 from the minireview shows a schematic depiction of γ-secretase and SPP

http://www.asbmb.org/assets/0/366/418/428/85528/85529/85530/c2de032a-daad-41e5-ba19-87a17bd26362.png

GxGD proteases are a family of intramembranous enzymes capable of hydrolyzing

  • the transmembrane domain of some integral membrane proteins.

The GxGD family is one of the three families of

  • intramembrane-cleaving proteases discovered so far (along with the rhomboid and site-2 protease) and
  • includes the γ-secretase and the signal peptide peptidase.

Although only recently discovered, a number of functions in human pathology and in numerous other biological processes

  • have been attributed to γ-secretase and SPP.

Taisuke Tomita and Takeshi Iwatsubo of the University of Tokyo highlighted the latest findings on the structure and function of γ-secretase and SPP
in a recent minireview in The Journal of Biological Chemistry.

  • γ-secretase is involved in cleaving the amyloid-β precursor protein, thus producing amyloid-β peptide,

the main component of senile plaques in Alzheimer’s disease patients’ brains. The complete structure of mammalian γ-secretase is not yet known; however,
Tomita and Iwatsubo note that biochemical analyses have revealed it to be a multisubunit protein complex.

  • Its catalytic subunit is presenilin, an aspartyl protease.

In vitro and in vivo functional and chemical biology analyses have revealed that

  • presenilin is a modulator and mandatory component of the γ-secretase–mediated cleavage of APP.

Genetic studies have identified three other components required for γ-secretase activity:

  1. nicastrin,
  2. anterior pharynx defective 1 and
  3. presenilin enhancer 2.

By coexpression of presenilin with the other three components, the authors managed to

  • reconstitute γ-secretase activity.

Tomita and Iwatsubo determined using the substituted cysteine accessibility method and by topological analyses, that

  • the catalytic aspartates are located at the center of the nine transmembrane domains of presenilin,
  • by revealing the exact location of the enzyme’s catalytic site.

The minireview also describes in detail the formerly enigmatic mechanism of γ-secretase mediated cleavage.

SPP, an enzyme that cleaves remnant signal peptides in the membrane

  • during the biogenesis of membrane proteins and
  • signal peptides from major histocompatibility complex type I,
  • also is involved in the maturation of proteins of the hepatitis C virus and GB virus B.

Bioinformatics methods have revealed in fruit flies and mammals four SPP-like proteins,

  • two of which are involved in immunological processes.

By using γ-secretase inhibitors and modulators, it has been confirmed

  • that SPP shares a similar GxGD active site and proteolytic activity with γ-secretase.

Upon purification of the human SPP protein with the baculovirus/Sf9 cell system,

  • single-particle analysis revealed further structural and functional details.

HLA targeting efficiency correlates with human T-cell response magnitude and with mortality from influenza A infection

From www.pnas.org –  Sep 3, 2013 4:24 PM

Experimental and computational evidence suggests that

  • HLAs preferentially bind conserved regions of viral proteins, a concept we term “targeting efficiency,” and that
  • this preference may provide improved clearance of infection in several viral systems.

To test this hypothesis, T-cell responses to A/H1N1 (2009) were measured from peripheral blood mononuclear cells obtained from a household cohort study
performed during the 2009–2010 influenza season. We found that HLA targeting efficiency scores significantly correlated with

  • IFN-γ enzyme-linked immunosorbent spot responses (P = 0.042, multiple regression).

A further population-based analysis found that the carriage frequencies of the alleles with the lowest targeting efficiencies, A*24,

  • were associated with pH1N1 mortality (r = 0.37, P = 0.031) and
  • are common in certain indigenous populations in which increased pH1N1 morbidity has been reported.

HLA efficiency scores and HLA use are associated with CD8 T-cell magnitude in humans after influenza infection.
The computational tools used in this study may be useful predictors of potential morbidity and

  • identify immunologic differences of new variant influenza strains
  • more accurately than evolutionary sequence comparisons.

Population-based studies of the relative frequency of these alleles in severe vs. mild influenza cases

  • might advance clinical practices for severe H1N1 infections among genetically susceptible populations.

Metabolomics in drug target discovery

J D Rabinowitz et al.

Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ.
Cold Spring Harbor Symposia on Quantitative Biology 11/2011; 76:235-46.
http://dx.doi.org:/10.1101/sqb.2011.76.010694 

Most diseases result in metabolic changes. In many cases, these changes play a causative role in disease progression. By identifying pathological metabolic changes,

  • metabolomics can point to potential new sites for therapeutic intervention.

Particularly promising enzymatic targets are those that

  • carry increased flux in the disease state.

Definitive assessment of flux requires the use of isotope tracers. Here we present techniques for

  • finding new drug targets using metabolomics and isotope tracers.

The utility of these methods is exemplified in the study of three different viral pathogens. For influenza A and herpes simplex virus,

  • metabolomic analysis of infected versus mock-infected cells revealed
  • dramatic concentration changes around the current antiviral target enzymes.

Similar analysis of human-cytomegalovirus-infected cells, however, found the greatest changes

  • in a region of metabolism unrelated to the current antiviral target.

Instead, it pointed to the tricarboxylic acid (TCA) cycle and

  • its efflux to feed fatty acid biosynthesis as a potential preferred target.

Isotope tracer studies revealed that cytomegalovirus greatly increases flux through

  • the key fatty acid metabolic enzyme acetyl-coenzyme A carboxylase.
  • Inhibition of this enzyme blocks human cytomegalovirus replication.

Examples where metabolomics has contributed to identification of anticancer drug targets are also discussed. Eventual proof of the value of

  • metabolomics as a drug target discovery strategy will be
  • successful clinical development of therapeutics hitting these new targets.

 Related References

Use of metabolic pathway flux information in targeted cancer drug design. Drug Discovery Today: Therapeutic Strategies 1:435-443, 2004.

Detection of resistance to imatinib by metabolic profiling: clinical and drug development implications. Am J Pharmacogenomics. 2005;5(5):293-302. Review. PMID: 16196499

Medicinal chemistry, metabolic profiling and drug target discovery: a role for metabolic profiling in reverse pharmacology and chemical genetics.
Mini Rev Med Chem.  2005 Jan;5(1):13-20. Review. PMID: 15638788 [PubMed – indexed for MEDLINE] Related citations

Development of Tracer-Based Metabolomics and its Implications for the Pharmaceutical Industry. Int J Pharm Med 2007; 21 (3): 217-224.

Use of metabolic pathway flux information in anticancer drug design. Ernst Schering Found Symp Proc. 2007;(4):189-203. Review. PMID: 18811058

Pharmacological targeting of glucagon and glucagon-like peptide 1 receptors has different effects on energy state and glucose homeostasis in diet-induced obese mice. J Pharmacol Exp Ther. 2011 Jul;338(1):70-81. http://dx.doi.org:/10.1124/jpet.111.179986. PMID: 21471191

Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the
[U-C(6)]-d-glucose tracer in mice. Metabolomics. 2009 Sep;5(3):336-345. PMID: 19718458

Metabolic Pathways as Targets for Drug Screening, Metabolomics, Dr Ute Roessner (Ed.), ISBN: 978-953-51-0046-1, InTech, Available from: http://www.intechopen.com/books/metabolomics/metabolic-pathways-as-targets-for-drug-screening

Iron regulates glucose homeostasis in liver and muscle via AMP-activated protein kinase in mice. FASEB J. 2013 Jul;27(7):2845-54.
http://dx.doi.org:/10.1096/fj.12-216929. PMID: 23515442

Metabolomics and systems pharmacology: why and how to model the human metabolic network for drug discovery

Drug Discov. Today 19 (2014), 171–182     http://dx.doi.org:/10.1016/j.drudis.2013.07.014

Highlights

  • We now have metabolic network models; the metabolome is represented by their nodes.
  • Metabolite levels are sensitive to changes in enzyme activities.
  • Drugs hitchhike on metabolite transporters to get into and out of cells.
  • The consensus network Recon2 represents the present state of the art, and has predictive power.
  • Constraint-based modelling relates network structure to metabolic fluxes.

Metabolism represents the ‘sharp end’ of systems biology, because changes in metabolite concentrations are

  • necessarily amplified relative to changes in the transcriptome, proteome and enzyme activities, which can be modulated by drugs.

To understand such behaviour, we therefore need (and increasingly have) reliable consensus (community) models of

  • the human metabolic network that include the important transporters.

Small molecule ‘drug’ transporters are in fact metabolite transporters, because

  • drugs bear structural similarities to metabolites known from the network reconstructions and
  • from measurements of the metabolome.

Recon2 represents the present state-of-the-art human metabolic network reconstruction; it can predict inter alia:

(i) the effects of inborn errors of metabolism;

(ii) which metabolites are exometabolites, and

(iii) how metabolism varies between tissues and cellular compartments.

However, even these qualitative network models are not yet complete. As our understanding improves

  • so do we recognise more clearly the need for a systems (poly)pharmacology.

Introduction – a systems biology approach to drug discovery

It is clearly not news that the productivity of the pharmaceutical industry has declined significantly during recent years

  • following an ‘inverse Moore’s Law’, Eroom’s Law, or
  • that many commentators, consider that the main cause of this is
  • because of an excessive focus on individual molecular target discovery rather than a more sensible strategy
  • based on a systems-level approach (Fig. 1).
drug discovery science

drug discovery science

Figure 1.

The change in drug discovery strategy from ‘classical’ function-first approaches (in which the assay of drug function was at the tissue or organism level),
with mechanistic studies potentially coming later, to more-recent target-based approaches where initial assays usually involve assessing the interactions
of drugs with specified (and often cloned, recombinant) proteins in vitro. In the latter cases, effects in vivo are assessed later, with concomitantly high levels of attrition.

Arguably the two chief hallmarks of the systems biology approach are:

(i) that we seek to make mathematical models of our systems iteratively or in parallel with well-designed ‘wet’ experiments, and
(ii) that we do not necessarily start with a hypothesis but measure as many things as possible (the ’omes) and

  • let the data tell us the hypothesis that best fits and describes them.

Although metabolism was once seen as something of a Cinderella subject,

  • there are fundamental reasons to do with the organisation of biochemical networks as
  • to why the metabol(om)ic level – now in fact seen as the ‘apogee’ of the ’omics trilogy –
  •  is indeed likely to be far more discriminating than are
  • changes in the transcriptome or proteome.

The next two subsections deal with these points and Fig. 2 summarises the paper in the form of a Mind Map.

metabolomics and systems pharmacology

metabolomics and systems pharmacology

http://ars.els-cdn.com/content/image/1-s2.0-S1359644613002481-gr2.jpg

Metabolic Disease Drug Discovery— “Hitting the Target” Is Easier Said Than Done

David E. Moller, et al.   http://dx.doi.org:/10.1016/j.cmet.2011.10.012

Despite the advent of new drug classes, the global epidemic of cardiometabolic disease has not abated. Continuing

  • unmet medical needs remain a major driver for new research.

Drug discovery approaches in this field have mirrored industry trends, leading to a recent

  • increase in the number of molecules entering development.

However, worrisome trends and newer hurdles are also apparent. The history of two newer drug classes—

  1. glucagon-like peptide-1 receptor agonists and
  2. dipeptidyl peptidase-4 inhibitors—

illustrates both progress and challenges. Future success requires that researchers learn from these experiences and

  • continue to explore and apply new technology platforms and research paradigms.

The global epidemic of obesity and diabetes continues to progress relentlessly. The International Diabetes Federation predicts an even greater diabetes burden (>430 million people afflicted) by 2030, which will disproportionately affect developing nations (International Diabetes Federation, 2011). Yet

  • existing drug classes for diabetes, obesity, and comorbid cardiovascular (CV) conditions have substantial limitations.

Currently available prescription drugs for treatment of hyperglycemia in patients with type 2 diabetes (Table 1) have notable shortcomings. In general,

Therefore, clinicians must often use combination therapy, adding additional agents over time. Ultimately many patients will need to use insulin—a therapeutic class first introduced in 1922. Most existing agents also have

  • issues around safety and tolerability as well as dosing convenience (which can impact patient compliance).

Pharmacometabolomics, also known as pharmacometabonomics, is a field which stems from metabolomics,

  • the quantification and analysis of metabolites produced by the body.

It refers to the direct measurement of metabolites in an individual’s bodily fluids, in order to

  • predict or evaluate the metabolism of pharmaceutical compounds, and
  • to better understand the pharmacokinetic profile of a drug.

Alternatively, pharmacometabolomics can be applied to measure metabolite levels

  • following the administration of a pharmaceutical compound, in order to
  • monitor the effects of the compound on certain metabolic pathways(pharmacodynamics).

This provides detailed mapping of drug effects on metabolism and

  • the pathways that are implicated in mechanism of variation of response to treatment.

In addition, the metabolic profile of an individual at baseline (metabotype) provides information about

  • how individuals respond to treatment and highlights heterogeneity within a disease state.

All three approaches require the quantification of metabolites found

relationship between -OMICS

relationship between -OMICS

http://upload.wikimedia.org/wikipedia/commons/thumb/e/eb/OMICS.png/350px-OMICS.png

Pharmacometabolomics is thought to provide information that

Looking at the characteristics of an individual down through these different levels of detail, there is an

  • increasingly more accurate prediction of a person’s ability to respond to a pharmaceutical compound.
  1. the genome, made up of 25 000 genes, can indicate possible errors in drug metabolism;
  2. the transcriptome, made up of 85,000 transcripts, can provide information about which genes important in metabolism are being actively transcribed;
  3. and the proteome, >10,000,000 members, depicts which proteins are active in the body to carry out these functions.

Pharmacometabolomics complements the omics with

  • direct measurement of the products of all of these reactions, but with perhaps a relatively
  • smaller number of members: that was initially projected to be approximately 2200 metabolites,

but could be a larger number when gut derived metabolites and xenobiotics are added to the list. Overall, the goal of pharmacometabolomics is

  • to more closely predict or assess the response of an individual to a pharmaceutical compound,
  • permitting continued treatment with the right drug or dosage
  • depending on the variations in their metabolism and ability to respond to treatment.

Pharmacometabolomic analyses, through the use of a metabolomics approach,

  • can provide a comprehensive and detailed metabolic profile or “metabolic fingerprint” for an individual patient.

Such metabolic profiles can provide a complete overview of individual metabolite or pathway alterations,

This approach can then be applied to the prediction of response to a pharmaceutical compound

  • by patients with a particular metabolic profile.

Pharmacometabolomic analyses of drug response are

Pharmacogenetics focuses on the identification of genetic variations (e.g. single-nucleotide polymorphisms)

  • within patients that may contribute to altered drug responses and overall outcome of a certain treatment.

The results of pharmacometabolomics analyses can act to “inform” or “direct”

  • pharmacogenetic analyses by correlating aberrant metabolite concentrations or metabolic pathways to potential alterations at the genetic level.

This concept has been established with two seminal publications from studies of antidepressants serotonin reuptake inhibitors

  • where metabolic signatures were able to define a pathway implicated in response to the antidepressant and
  • that lead to identification of genetic variants within a key gene
  • within the highlighted pathway as being implicated in variation in response.

These genetic variants were not identified through genetic analysis alone and hence

  • illustrated how metabolomics can guide and inform genetic data.

en.wikipedia.org/wiki/Pharmacometabolomics

Benznidazole Biotransformation and Multiple Targets in Trypanosoma cruzi Revealed by Metabolomics

Andrea Trochine, Darren J. Creek, Paula Faral-Tello, Michael P. Barrett, Carlos Robello
Published: May 22, 2014   http://dx.doi.org:/10.1371/journal.pntd.0002844

The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi,

  • involves administration of benznidazole (Bzn).

Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active. We used a

  • non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.

Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols

  1. trypanothione,
  2. homotrypanothione and
  3. cysteine

were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment.

These metabolites included reduction products, fragments and covalent adducts of reduced Bzn

  • linked to each of the major low molecular weight thiols:
  1. trypanothione,
  2. glutathione,
  3. g-glutamylcysteine,
  4. glutathionylspermidine,
  5. cysteine and
  6. ovothiol A.

Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI,

  • were found within the parasites,
  • but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.

Our data is indicative of a major role of the

  • thiol binding capacity of Bzn reduction products
  • in the mechanism of Bzn toxicity against T. cruzi.

 

 

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